Intracellular Uncoating of Type 5 Adenovirus Deoxyribonucleic Acid

Abstract

Highly purified, "P-labeled type 5 adenovirus was employed to study "uncoating" of viral deoxyribonucleic acid (DNA)-defined as the development of sensitivity to deoxyribonuclease. Viral infectivity and radioactivity adsorbed to KB cells at the same rate, and significant amounts of "2P did not elute from cells throughout the eclipse period. Kinetic studies of viral penetration, eclipse of infectivity, and uncoating of viral DNA indicated that the three events were closely related temporally, that the rates of each were similar, and that they were completed within 60 to 90 min after infection. Viral penetration, eclipse, and uncoating proceeded normally under conditions which blocked protein synthesis, but they did not occur at 0 to 4 C. Neither viral DNA nor viral protein was degraded to acid-soluble material during the eclipse period. The nature of adenovirus DNA was studied after it was converted intracellularly from deoxyribonuclease-resistant to deoxyribonucleasesusceptible. Intact virions centrifuged in sucrose gradients had a sedimentation coefficient of approximately 800, and viral DNA sedimented as a particle of about 30S. Infection of KB cells with purified "2P-labeled virus yielded deoxyribonucleasesusceptible viral nucleic acid which was in particles with sedimentation coefficients of 350 to 450S, i.e., greater than 10 times faster than DNA obtained from purified virions which had been disrupted by exposure to pH 10.5. When the DNA from disrupted virions was mixed with cell lysates, its sedimentation characteristics were essentially unchanged by the presence of cellular material.

Early Events of Virus-Cell Interaction in an Adenovirus System

“…deoxyribonuclease sensitivity of the product, the iles, first-order model would also fit, at early times, unit with a half-life of 15 min. This also agrees with bot-the rapid uncoating observed by Philipson with in a adenovirus type 2 (21) and by Lawrence and 4 of Ginsberg with adenovirus type 5 (12).…”

“…Lawrence and Ginsberg also observed that about 40% of the 32p from labeled adenovirustype 5 gains entry, after cell attachment, into what was described as a crude nuclear fraction of KB cells (12). Hochberg and Becker have re-ported the uncoating and entry of labeled DNA from herpesvirus into BSC cell nuclei (7).…”

“…The interaction of virion and cell can be examined in considerable detail with the human adenoviruses, because adenovirus can be readily labeled with isotopes (10,12,21,24), purified and maintained in relatively stable infective pools (3,21). Attachment of labeled virus to cultured human cells is also rapid and efficient (12,21). In addition, there is available a wealth of background information on virion morphology, chemical composition, and also on the specific biological activities of some of the individual virion proteins (for reviews, see 17 and 26).…”

“…It has been shown that much of the deoxyribonucleic acid (DNA) of cell-attached type 2 or 5 adenovirions becomes accessible to digestion by deoxyribonuclease (E.C. 3.1.4.5) within less than about 30 min at 37 C and that the direct product of this "uncoating" is not naked viral DNA (12,21). Furthermore, Sussenbach has shown that one uncoating product of adenovirus type 5 has I Recipient of special Public Health Service research fellowship award SF3-AI-6864 from the National Institute of Allergy and Infectious Diseases.…”

Hexon peptides of type 2, 3, and 5 adenoviruses and their relationship to hexon structure

“…The prototype strains of adenovirus types 2, 3, and 5, which had been plaque purified three times, were used. After infecting monolayers of KB cells, virus was quantitated by plaque (12) or fluorescent focus assay (5).…”