p53-Dependent apoptosis suppresses tumor growth and progression in vivo

Publisher Summary It is clear from the experimental data that there are some similarities in RNA replication for all eukaryotic positive-stranded RNA viruses—that is, the mechanism of polymerization of the nucleotides is probably similar for all. It is noteworthy that all mechanisms appear to utilize host membranes as a site of replication. Membranes appear to function not only as a way of compartmentalizing virus RNA replication but also appear to have a central role in the organization and functioning of the replication complex, and further studies in this area are needed. Within virus supergroups, similarities are evident between animal and plant viruses—for example, in the nature and arrangements of replication genes and in sequence similarities of functional domains. However, it is also clear that there has been considerable divergence, even within supergroups. For example, the animal alpha-viruses have evolved to encode proteinases which play a central controlling function in the replication cycle, whereas this is not common in the plant alpha-like viruses and even when it occurs, as in the tymoviruses, the strategies that have evolved appear to be significantly different. Some of the divergence could be host-dependent and the increasing interest in the role of host proteins in replication should be fruitful in revealing how different systems have evolved. Finally, there are virus supergroups that appear to have no close relatives between animals and plants, such as the animal coronavirus-like supergroup and the plant carmo-like supergroup.

Comparison of The Replication of Positive-Stranded Rna Viruses of Plants and Animals

https://www.cell.com/structure/pdf/S0969-2126(97)00261-X.pdf

Structure of the RNA-dependent RNA polymerase of poliovirus

The transfer of genetic information into mouse embryos to stably alter the genetic constitution of mice is affording new insights into and opportunities in a wide variety of biological problems. Higher eukaryotes are composed of many interacting cells and organs. The properties of individual cell systems are often discernible only by studying natural or induced disruptions in their functions. Transgenic mice represent a new form of perturbation analysis whereby the selective expression of novel or altered genes can be used to perturb complex systems in ways that are informative about their development, their functions, and their malfunctions. The utility of this strategy is illustrated by recent research into immunological self-tolerance, oncogenes and cancer, and development.

http://science.sciencemag.org/content/sci/246/4935/1265.full.pdf

Transgenic mice as probes into complex systems

Myocarditis is a cardiac disease associated with inflammation and injury of the myocardium. Several viruses have been associated with myocarditis in humans. However, coxsackievirus B3 is still considered the dominant etiological agent. The observed pathology in viral myocarditis is a result of cooperation or teamwork between viral processes and host immune responses at various stages of disease. Both innate and adaptive immune responses are crucial determinants of the severity of myocardial damage, and contribute to the development of chronic myocarditis and dilated cardiomyopathy following acute viral myocarditis. Advances in genomics and proteomics, and in the use of informatics and biostatistics, are allowing unbiased initial evaluations that can be the basis for testable hypotheses about virus pathogenesis and new therapies.

Molecular biology and pathogenesis of viral myocarditis

A poliovirus-specific RNA-dependent RNA polymerase was isolated from a cytoplasmic extract of infected HeLa cells and was shown to copurify with a single virus-specific protein. The polymerase was isolated from cells labeled with [35S]-methionine and was fractionated from other soluble cytoplasmic proteins by ammonium sulfate precipitation, phosphocellulose chromatography, gel filtration on Sephacryl S-200, and chromatography on hydroxylapatite. The activity of the enzyme was measured by using either polyadenylic acid or poliovirion RNA as a template in the presence of an oligouridylic acid primer. A single virus-specific protein that had an apparent molecular weight of 63,000 (p63) was found to copurify with this activity. Host-coded proteins were present in reduced molar amounts relative to p63. Noncapsid viral protein 2 (NCVP2) and other viral proteins were clearly separated from p63 by gel filtration on Sephacryl S-200. Polymerase activity coeluted from the column precisely with p63. NCVP2 was totally inactive as an RNA polymerase and did not stimulate the polymerase activity of p63. The purified enzyme sedimented at about 4S on a glycerol gradient and thus appeared to be a monomer of p63. Two-dimensional gel electrophoresis of the polymerase protein indicated that it had an isoelectric point of about 7.5. Thus, the viral polypeptide, p63, as defined by the above physical parameters, is an RNA-dependent RNA polymerase that can copy poliovirion RNA when oligouridylic acid is used as a primer.

Identification of poliovirus polypeptide P63 as a soluble RNA-dependent RNA polymerase.

A soluble RNA-dependent RNA polymerase was isolated from poliovirus-infected HeLa cells and was shown to copy poliovirus RNA in vitro. The enzyme was purified from a 200,000-X-g supernatant of a cytoplasmic extract of infected cells. The activity of the enzyme was measured throughout the purification by using a polyadenylic acid template and oligouridylic acid primer. The enzyme was partially purified by ammonium sulfate precipitation, glycerol gradient centrifugation, and phosphocellulose chromatography. The polymerase precipitated in a 35% saturated solution of ammonium sulfate, sedimented at about 7S on a glycerol gradient, and eluted from phosphocellulose with 0.15 M KC1. The polymerase was purified about 40-fold and was shown to be totally dependent on exogenous RNA for activity and relatively free of contaminating nuclease. The partially purified polymerase was able to use purified polio virion RNA as well as a template. Under the reaction conditions used, the polymerase required an oligouridylic acid primer and all four ribonucleside triphosphates for activity. The optimum ratio of oligouridylic acid molecules to poliovirus RNA molecules for priming activity was about 16:1. A nearest-neighbor analysis of the in vitro RNA product shows it to be heteropolymeric. Annealing the in vitro product with poliovirus RNA product shows it to be heteropolymeric. Annealing the in vitro product with poliovirus RNA rendered it resistant to RNase digestion, thus suggesting that the product RNA was complementary to the virion RNA template.

Isolation of a soluble and template-dependent poliovirus RNA polymerase that copies virion RNA in vitro.

A line of transgenic mice containing the simian virus 40 (SV40) large tumor antigen gene under the control of the viral enhancer-promoter expressed this viral protein in the brains of these mice within the first 2 weeks after birth. Multiple foci of anaplastic cells formed in the choroid plexuses of these mice at 36 to 41 days after birth, and normal tissue coexisted with these transformed foci. Immunoperoxidase staining to detect the SV40 T antigen showed tumor-specific expression of nuclear T antigen at late times in tumor development, approximately 90 to 100 days and thereafter. The level of SV40 T antigen, on a per cell basis, appeared to be lower in the great majority of choroid plexus cells at earlier times in tumor development. These results suggest that low levels of tumor antigen (14 to 36 days) are present before detectable pathology (36 to 41 days) and the level of T antigen per cell is higher in rapidly growing late-stage tumors (older than 90 days).

T A Van Dyke

Papers

Identification of poliovirus polypeptide P63 as a soluble RNA-dependent RNA polymerase.

Isolation of a soluble and template-dependent poliovirus RNA polymerase that copies virion RNA in vitro.

Relationship between simian virus 40 large tumor antigen expression and tumor formation in transgenic mice.

Relationship between Simian Virus 40LargeTumorAntigen Expression andTumorFormation inTransgenic Mice