In vitro translation of adenovirus type 12-specific mRNA isolated from infected and transformed cells

Abstract

The early and late gene products of human adenovirus type 12 (Ad12), as well as the viral proteins synthesized in an Ad12-transformed cell line, were identified by translation of viral mRNA in an in vitro protein-synthesizing system. Cytoplasmic RNA was isolated from permissive KB or nonpermissive BHK cells infected with Ad12 and from Ad12-transformed HA12/7 cells. Virus-specific RNA was selected by hybridization to Ad12 DNA covalently bound to cellulose. Viral RNA was then translated in a fractionated rabbit reticulocyte cell-free system or in wheat germ S-30 extracts. The proteins synthesized were characterized by immunoprecipitation and subsequent electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. RNA prepared from KB cells late after infection with Ad12 elicited the synthesis of most of the structural polypeptides of the virion and at least two presumably nonstructural Ad12 proteins. When viral RNA isolated early after infection of KB cells with Ad12 was translated in vitro, 10 polypeptides were observed: E-68K, E-50K, E-42K, E-39K, E-34K, E-21K, E-19K, E-13K, E-12K, and E-10K. Ad12-specific RNA was also isolated from the Ad12-transformed hamster cell line HA12/7, which contains several copies of the Ad12 genome integrated in the host genome. The RNA codes for at least seven polypeptides with molecular weights very similar to those of the early viral proteins.

Nucleotide sequence of human adenovirus type 12 DNA: comparative functional analysis

“…Below the figure a scale in nucleotides serves as a frame of reference. functions on the genome by previously published DNA-RNA hybridization and cell-free translation experiments (19,20,30).…”

Defect of adenovirus type 12 replication in hamster cells: absence of transcription of viral virus-associated and L1 RNAs

“…It is concluded that the protection experiments performed in this study allowed the detection of VA RNAs in productively infected cells. In the abortively Adl2-infected BHK-21 cells, Adl2-specific VA RNA was not produced, even though most of the El functions of Adl2 DNA were transcribed (10,16). It is conceivable that in addition to the ElA functions (3,12), host cell functions are required to activate the polymerase III-mediated transcription of the Adl2-specific VA RNA.…”

“…There is a total block of Adl2 DNA replication in this host system (7,9,11), although virions are taken up into the cytoplasm and viral DNA enters the nucleus and can be integrated into the host genome (6,8). In hamster cells, most of the early viral functions are transcribed into mRNA (16)(17)(18) which can be expressed in a cell-free transcription system (10). In contrast, transcription of the late Adl2 genes in hamster cells cannot be detected (13,14,17).…”

Adenovirus types 2 and 5 functions elicit replication and late expression of adenovirus type 12 DNA in hamster cells

“…It should be noted, however, that the early functions of Adl2 DNA can be efficiently expressed in hamster cells (14,15,23,28,34). Hence, the trans-activation by Ad2 or Ad5 functions appears to be required mainly for the expression of the late Adl2 functions and for the replication of Adl2 DNA.…”

“…Adl2 virions do infect hamster cells; the viral DNA reaches the nucleus, and a portion of the parental viral DNA molecules is integrated into the host genome (9,11). The early functions of Adl2 DNA are expressed (27,28,30), whereas RNA sequences homologous to the late Adl2 genes cannot be detected in hamster cells (14,28). It could be demonstrated by in vitro translation of hybrid-selected RNAs that, in Adl2-infected BHK-21 cells, all of the early viral mRNAs are synthesized as in productively infected cells, except for a 34,000molecular-weight (34K) protein from the ElA region of Adl2 DNA (14).…”