Donald F. Summers

Effects on host cell metabolism following synchronous infection with poliovirus.

Abstract The inhibition of host cell protein synthesis by poliovirus was measured in synchronously infected cells. The inhibition was manifested only after a period of protein synthesis following virus adsorption. The rate of inhibition of host protein synthesis, at both high and low multiplicities of virus, was only slightly affected by concentrations of guanidine which prevent viral RNA replication. This implies that the inhibition of host protein synthesis can be effected by the initial infecting virus. To test that the inhibition is not a result of the attaching of virus particles, the experiments were repeated with poliovirus that had been inactivated by a factor of 10 3 by UV. These particles had no effect on host protein synthesis. Synchronously infected cells were also used to study the stimulation of incorporation of choline into acid-precipitable material which is caused by poliovirus. The stimulation is evident only after extensive viral RNA replication has taken place. When viral RNA replication is prevented by the addition of guanidine the stimulation of choline incorporation is completely suppressed at low virus input multiplicities and reduced by about two-thirds at high multiplicity.

Composition of artificially produced and naturally occurring empty capsids of poliovirus type 1.

The polypeptide composition of the empty capsids of poliovirus type 1 represented either by the 73 S component isolated from sucrose gradients or by the top component from CsCl gradients is different from that ot the whole infectious virion. One protein (VP4) is absent from the empty capsids, another (VP2) is lower in relative amount, while an additional protein (NCVP6), not present in purified virions, is found in large amounts. Artificial production of 73 S particles by borate buffer, pH 10.5, treatment of purified virions results in removal of one protein component (VP4) and the RNA of the virus. The observed differences in composition of these virus-specific particles are discussed with regard to their possible role in the architecture and the assembly of the virion.

Protein synthesis in vesicular stomatitis virus-infected HeLa cells

Abstract A method is presented for maximizing the amount of radioactive precursors incorporated into vesicular stomatitis virus (VSV) grown in suspension cultures of HeLa S3 cells. By varying the input multiplicity of infection, different relative rates of VSV protein synthesis and of host protein synthesis are found. All the virus-specific polypeptides resolved by polyacrylamide gel analysis are synthesized in the same relative proportions throughout the infectious cycle; the predominant species synthesized correspond to the polypeptides of the purified virion, and no evidence is found for a large polypeptide precursor to any of the virus-specific polypeptides. The molecular weights of the capsid polypeptides are 180,000, 67,000, 52,000, 40,000, and 25,000. As many as 9 additional polypeptides are seen in infected cells. The molecular weight of the RNA from purified virions is 3.6 × 106 daltons under both native and denaturing conditions. This size is sufficient to account for the total mass of polypeptides synthesized in the infected cell, provided that polypeptides larger than 70,000 daltons are disregarded.

Evidence for differences in size and composition of the poliovirus-specific polypeptides in infected HeLa cells☆

Abstract The poliovirus specific capsid and noncapsid proteins have been shown by use of a radioisotopic method to have differences in amino acid composition. Differences in size were demonstrated by sucrose gradient analysis, molecular sieve chromatography and acrylamide gel electrophoresis. Molecular weights of all the virus-specific polypeptides were also obtained by the latter method; the molecular weights ranged from 79,000 to 5000. The total combined molecular weights of the fourteen virus-specific proteins is approximately 500,000 daltons. This mass greatly exceeds the 200,000 daltons of total protein expected to be encoded in the poliovirus genome which supports our previous evidence for large precursor proteins.

Polysomal ribonucleic acid of vesicular stomatitis virus-infected HeLa cells

Abstract The vesicular stomatitis virus-specific messenger RNA fraction of infected HeLa cell polysomes has been identified by (1) its removal from the polysome region of sucrose gradients by EDTA treatment, and (2) its sensitivity to ribonuclease. This mRNA fraction anneals to virion RNA and is a collection of several species of small RNA molecules of the approximate size distribution appropriate to code for the virus-specific polypeptides found in infected cells. The base composition of the mRNA region of sucrose gradients is lower in U and higher and A than would be predicted for RNA complementary to total virion RNA. This finding, along with labeling kinetics and ribonuclease resistance of structures found in the polysome region of sucrose gradients, gives rise to two possible working models for the mechanism of vesicular stomatitis virus mRNA synthesis.

In vitro assembly of poliovirus-related particles☆

Abstract A viral fraction has been isolated from poliovirus-infected HeLa cells which sediments at 10 S and which contains virus-specific polypeptides closely resembling those of 73 S empty capsids. In the presence of infected cell extracts, the 10 S material is assembled to a 73 S product indistinguishable from natural empty capsids by sedimentation behavior or by polypeptide analysis with gel electrophoresis. Kinetic experiments suggest that the 10 S and 73 S components are sequential precursors of the complete virion.

Soluble RNA polymerase complex from poliovirus-infected HeLa cells

Abstract A stable, soluble poliovirus RNA-polymerase complex has been isolated from particulate structures in poliovirus-infected HeLa cells with an anionic-nonionic detergent mixture. The complex sediments in a sucrose gradient with a sedimentation constant of approximately 70 S. The isolated enzyme activity is proportional to the rate of viral RNA synthesis in vivo throughout the infectious cycle. Inhibition of protein synthesis with cycloheximide during time of maximal virus replication results in a rapid fall of RNA synthesis and a proportional exponential decrease in isolated polymerase complex activity, the decay occurring with a t 1 2 = 15 min . Properties of the in vitro polymerase reaction are described. The products synthesized by the enzyme complex include RNase-resistant material sedimenting at 20 S, and RNase-sensitive material that cosediments with RNA extracted from purified virions at 35 S. A large fraction of the RNase-resistant material becomes RNase-sensitive after heating for 10 min at 45 °; the remainder requires heating at temperatures greater than 100 °. The polymerase complex has been purified approximately 85-fold. Activity is independent of exogenous polio RNA template.

In vitro protein synthetic activity of membrane-bound poliovirus polyribosomes.

Abstract In vitro protein synthesis activity of subcellular fractions of polio-infected and uninfected HeLa cells was examined in cytoplasmic extracts, cytoplasmic pellets, membrane-bound polyribosomes, and free polyribosomes. Membrane-bound polyribosomes had five times greater relative activity than free polysomes in vitro. The polio specific products of infected cell fractions were examined by sodium dodecyl sulfate-acrylamide gel electrophoresis, and it was found that in vitro systems were variously defective in the cleavage of large poliovirus precursor polypeptides. Membrane-bound polyribosomes synthesized but did not extensively cleave a large protein of the size to be expected from uninterrupted translation of the entire poliovirus genome. Crude cytoplasmic extracts and pellets synthesized peptides of intermediate size suggesting that they can perform some precursor cleavage.

The decrease in size and synthetic activity of poliovirus polysomes late in the infectious cycle

Abstract Poliovirus-induced polysomes in infected HeLa cells have been examined during the course of the infectious cycle with respect to (1) sedimentation behavior, (2) the average number of host cell ribosomes per viral messenger RNA molecule, and (3) the spectrum of capsid and noncapsid virus specific polypeptide chains being synthesized. It was found that the polysomes are large (380 S) and indistinguishable in size during most of the RNA replicative cycle. Late in infection, however, the average size of the virus polysomes decreases. The average number of ribosomes in the largest polysomes is about 35 per viral mRNA molecule while in the smaller late-cycle polysomes, which are about 200 S, there are still an average of 20 ribosomes. Furthermore, the rate of protein synthesis per ribosome is considerably decreased in the late cycle polysomes, although the same spectrum of viral proteins is made throughout the replicative cycle.

Evidence for ambiguity in the posttranslational cleavage of poliovirus proteins.

Abstract The molecular weights (estimated from mobilities in acrylamide gels) of the proteins of ts + and most temperature-sensitive (ts) strains of type 1 poliovirus correspond closely with those of their ancestral strain Mahoney. In one mutant strain, ts -89, the large precursor protein, NCVP 1, was cleaved more slowly at the restrictive temperature than that of ts + . The ts and ts + proteins differed from those of Mahoney in that the ts + structural protein VP 1B (31,000 daltons) was not detected in Mahoney virions or infected cell cytoplasms, while a normal Mahoney structural protein VP 1A (36,000 daltons) was absent from ts + and all ts virions except ts -2. Peptide analyses of proteins of ts + , several ts mutants and Mahoney virus did not reveal major differences and suggest that the primary sequences of the proteins of these strains do not differ extensively. Altogether, seven different structural proteins have been distinguished so far. As these exceed the probable translational capacity of the virus genome for such proteins, it is proposed that there are multiple cleavage sites in the precursor protein(s) and that these are used in varying combinations in different viral strains.