A method for determining the fraction of the viral genome transcribed during infection and its application to adenovirus-infected cells.

Abstract

Human adenovirus (Ad) DNA has a molecular weight of 23 X 106 daltons,1 thus containing sufficient information to code for 23-46 proteins. The viral capsid consists of about ten polypeptides2 that account for only a fraction of the number of different proteins possible. It is possible to estimate the extent to which this genetic information is utilized during lytic infection by (1) determining how many viral-coded proteins are synthesized in the infected cells as has been done with poliovirus-infected cells,' (2) studying conditional lethal virus mutants,4 or (3) determining what fraction of the viral gen.ome is transcribed to virus-specific messenger RNA (mRNA) molecules. We have chosen the third approach, utilizing a combination DNA-RNA (step 1) and DNA-DNA (step 2) hybridization procedure. In step 1, viral DNA immobilized on membrane filters is hybridized with unlabeled virus-specific RNA to saturate complementary DNA regions. In step 2, the presaturated viral DNA is further annealed with sheared, denatured, labeled, homologous viral DNA to determine the fraction of viral DNA not hybridized with RNA. The amount of labeled viral DNA bound is inversely related to the fraction of the genome transcribed. In this paper, we present the details of this procedure and the analysis of the fraction of Ad 2 viral genome transcribed during infection. 'xperimental Procedures.--Viral DNA: Suspension cultures of KB cells were grown in Eagle's minimum essential medium' containing 5% horse serum. The growth and purification of Ad 2 and the isolation of viral DNA have been described.6 Viral DNA was further purified by CsCl density gradient centrifugation. Viral [PI'] DNA was obtained from purified Ad 2 labeled with [P32] orthophosphate. Virus-specific RNA: Sujspension cultures of KB cells at 2-3 X 106 cells/ml were infected with Ad 2 (strain 38-2) at an input multiplicity of 100 PFIJ/cell. After 1 hr of adsorption at 370, the cell suspension was diluted in growth medium to a density of 2-3 X 1.0' cells/ml and incubated at 370 for an additional 17 hr. Cells were collected by centrifugation and RNA was extracted from the cell pellet by the hot phenol-sodium dodecyl sulfate (SDS) method,7 followed by a DNase treatment,8 a second hot phenol-SDS extraction,9 ethanol precipitation,9 and ether extraction.8 RNA was dialyzed against five changes of 0.1 X SSC (SSC == 0.15 M NaCl-0.015 M Na3 citrate) for 2 days. [H3] RNA was prepared from infected cells labeled with [HI] uridine (1 Mc/ml, 20 c/mM) for 30 min from 18 to 18.5 hr after infection and was purified by the procedure described above. DNA-RNA and DNA-DNA hybridization: In step 1, nitrocellulose membrane filters (Schleicher and Schuell, 136) containing immobilized Ad 2 DNA were incubated with unlabeled RNA from Ad 2-infected cells or from uninfected cells in 2 X SSC for 20 hr at 660.10=12 One set of filters, after treatment with RNase, was washed with 2 X SSC and incubated with sonicated, heat-denatured Ad 2 [P32] DNA in 2 X SSC containing 0.1% SDS for 20 hr at 60012, 13 (step 2). Filters were processed as described by Warnaar and Cohen,'4 and the extent of DNA-DNA hybridization was determined by counting bound [P32] DNA in a liquid scintillation counter. In order to estimate the completeness of