Components of Adenovirus-Simian Virus 40 Hybrid Viruses

Abstract

The I)NA and protein components of the adenovirus 7-simian virus (SV)40 hybrid virus "E46+" have been investigated. The Ad.7-SV40 hybrid DNA genome could be distinguished from nonhybrid Ad. 7 genome on the basis of its molecular weight in an alkaline sucrose gradient. The hybrid genome could also be separated from nonhybrid adenovirus 2 DNA when purified transcapsidant adenovirus 2+t7 (Ad.7-SV40 DNA within an Ad.2 capsid) was studied. MATERIALS AND METHODS VirusesThe E46+ strain of Ad.7-8V40 hybrid virus and its propagation in AGMIK and human embryonic kidney cells have been described in detail (2, 5). Adenovirus 2 + t7, a transcapsidant of E46+ in which the Ad.7-SV40 genome is encapsidated in an adenovirus type 2 coat (10), was received from Dr. Wallace Rowe. Both E46+ and Ad.2+t7 are mixtures of hybrid and nonhybrid virions because of the defective nature of the hybrid particles. E46-, the nonhybrid Ad.7 counterpart of E46+ derived by plaque l)urification, has also been described (9). All virus stocks were found to be free of adeno-associated viruses by serologic tests.The virus stocks used in these experiments were grown in human embryonic kidney cells obtained from Flow Laboratories (Bethesda, Md.), and were purified by published methods (11). The virus yields were titered by immunofluorescence and plaque assays as previously described (9,12

Genetic Evidence for a Temperature-Sensitive Lesion in the Adenovirus 7 Region of the PARA Genome

“…Phenotypic mixing of adenovirus capsids has been reported [26], but such mixing was not detected when the polypeptide components of purified PARA populations were analyzed by PAGE [9]. This technique would only detect components with altered molecular weights, however.…”

“…PARA-Ad 7 can induce SV40-typc tumors in vivo; it is highly oncogenic, whereas the parental Ad 7 is very weakly oncogenic in newborn hamsters [6,7], PARA also ex presses the SV40 function of enhancing human adenovirus replication in cells of simian origin [8]. Electron microscopic and nucleic acid hybridization data suggest that the hybrid virus genome contains ~60% of an intact SV40 genome and ~85% of an Ad 7 genome [9][10][11], Few functions have been recognized for the Ad 7 portion of the hybrid genome, partially due to the fact that the hybrid virus stocks always contain an excess of nonhybrid helper adenovirus [1]. Ad 7 T antigen has been detected in tumors induced by a PARA-Ad 12 transcapsidant population [12,13], No Ad 7 capsid (V) antigens have been detected in cells infected with transcapsid ant populations of PARA.…”

Synthesis of Viral Ribonucleic Acid During Restricted Adenovirus Infection

“…The titer of adenovirus stocks was determined by plaque assay on HEK monolayers (4), and that of SV40 stocks by plaque titration on AGMK or CV-1 monolayers. Infection of KB cell suspensions has been previously described (2).…”

“…Early adenovirus mRNA was prepared by a modification of the method of Fujinaga and Green (8). A suspension culture of 2 X 108 KB cells was concentrated and infected with Ad7 as described previously (2), except that cytosine arabinoside at a concentration of 20 mtg/ml (Sigma, St. Louis, Mo.) was added to the medium to prevent DNA synthesis.…”

“…Ad7 Complementary RNA (cRNA) and uninfected cell RNA. The preparation of Ad7 cRNA from Ad7 DNA in vitro with Escherichia coli polymerase and [3H]nucleotide triphosphates has been described (2). The preparation of RNA from uninfected CV-1 and uninfected KB cells was done as described above, except that the cells were mock infected with medium only.…”