Heschel J. Raskas

Biochemical studies on adenovirus multiplication. XVII. Ribosome synthesis in uninfected and infected KB cells.

Abstract Synthesis of 18 S and 28 S ribosomal RNA decreases in KB cells infected with adenovirus type 2 (Ad 2). At late times (18–20 hours) after infection the rate of entry of label into new ribosomes, as determined by analysis of total cellular RNA, polysomes, or free 45 S ribosome subunits is 20% the rate in uninfected cells. The ribosomes made in infected cells enter polysomes and are present in polysomes and free 45 S subunits in the same ratio as newly made ribosomes in uninfected cells. RNA labeled at 19 hours after infection was isolated from the polysome and free 45 S ribosome subunit regions of sucrose gradients and annealed with viral DNA. After a 90-min labeling period, 30% and 10% of the label in the polysome and 45 S subunit fractions hybridizes with Ad 2 DNA. Sucrose gradient analysis and hybridization studies of the RNA labeled 19 hours after infection indicate that ribosomal RNA and viral mRNA continue to enter the ribosome fractions at a constant ratio.

Thermosensitive events in the replication of adenovirus type 2 at 42

Abstract The production of infectious adenovirus type 2 is reduced more than 99% when infected KB suspension cultures are maintained at 42°. The abortive event(s) at 42° occur late after infection, for cultures held at 42° for 12 hr and then shifted to 37° yield normal amounts of infectious virus. Although 31 S viral DNA and virion polypeptides are synthesized at 42°, no 800 S viral particles are formed. The failure to produce virus at the elevated temperature can be attributed to lack of capsomere assembly. Although infected cultures incubated at 42° accumulate large amounts of structural viral polypeptides, formation of hexon capsomeres is reduced 99% and penton and fiber assembly are diminished more than 90%.

Partial Purification of Intracellular Murine Sarcoma-Leukemia Virus RNA Species by Membrane Filtration

Both 35S and 20S viral RNA species from transformed rat cells which produce the murine sarcoma-leukemia virus complex were retained on nitrocellulose membrane filters, yielding a 33-fold purification

Surface changes of human cells productively infected with human adenoviruses.

Abstract Productive infection of human embryonic kidney (HEK) cells with a group C adenovirus (type 2) resulted in surface changes that were detected as increased agglutination in the presence of concanavalin A (Con A). Agglutination increased as the infection progressed, with maximum response to Con A occurring by 48 hr. Treatment with cytosine arabinoside either for the entire infection or beginning 8 hr after infection prevented the surface alterations. In contrast to the agglutination produced by adenovirus 2, infection with a group A adenovirus (type 12) did not cause a changed response to Con A. However, infection with two cytocidal mutants derived from adenovirus 12 resulted in membrane changes detectable by Con A. These findings demonstrate that specific viral gene functions are required for the surface changes occurring during productive infection of adenoviruses. Since the cytocidal mutants are nononcogenic, the ability to produce surface changes during productive infection seems to be unrelated to the oncogenicity of the virus.

DNA–RNA and DNA–DNA Hybridization in Virus Research

Publisher Summary This chapter describes detailed methods and basic parameters of DNA-RNA and DNA-DNA hybridizations on nitrocellulose membranes. Nucleic acid hybridization refers to the specific interaction in vitro between two polynucleotide sequences by complementary base pairing. Increasing use of nucleic acid hybridization procedures in virology has made possible the direct studies of viral nucleic acid metabolism in virus-cell systems. With the precise hybridization methods, it is possible to study the metabolism of viral nucleic acid molecules and compare the sequence relationship of different viral molecules in any system. Hybrid formation between viral DNA and labeled RNA from infected or transformed cells provides a specific means for the identification, quantitation, and characterization of virus-specific RNA. The DNA-RNA procedure is based on the method of D. Gillespie and S. Spiegelman, and the DNA-DNA procedure was derived from the procedure of S. O. Warnaar and A. J. Cohen. Both these techniques are now being used extensively, with some modifications, for the study of nucleic acids of animal virus origin.