Three new virus-specific low molecular RNAs in adenovirus type 2 infected KB cells.

With the use of alkyl agarose columns which separate proteins according to their lipophilic affinities, a marked correlation has been uncovered between this parameter of proteins and their degradation rates $\text{in}$$\text{vivo}$. The possible relationship between these observations and analogous ones with other properties of proteins and their implications in regard to the mechanism of the turnover process are discussed.     

Identification of double-stranded virus-specific ribonucleic acid in KB cells infected with type 2 adenovirus

Using agarose gel chromatography, RNA with characteristics of double-stranded molecules was isolated from KB cells infected with type 2 adenovirus. Results of studies measuring its susceptibility to a number of nuclease activities were consistent with its identification as double-stranded RNA. The material was resistant to digestion by both pancreatic and T1 ribonuclease and by pancreatic deoxyribonuclease; and after heating at 100°C, it was much more sensitive to ribonuclease hydrolysis. Moreover, nearly 30% of the RNA formed a ribonuclease-resistant hybrid when denatured and incubated with purified type 2 adenovirus DNA, indicating that at least a portion of the double-stranded RNA in infected KB cells was transcribed from the viral genome.     

Transcription of the genome of adenovirus type 12. Viral mRNA in productively infected KB cells.

In human KB cells productively infected with adenovirus type 12, viral DNA replication starts between 12 and 14h postinfection. Virus-specific, polysome-associated mRNA was investigated early (6-8h) and late (26-28h) after infection. Most of the viral mRNA was polyadenylated and accounted for 0.46% and 24.1% of the mRNA synthesized early and late postinfection, respectively. The viral-specific mRNA isolated both early and late after infection falls into several distinct size-classes, ranging in molecular weights between 0.3X10(6) and 1.5X10(6) for the early RNA and between 0.6X10(6) and 2.3X10(6) for the RNA synthesized late in the infection.     

Stabilities and interrelations of multiple species of human adenovirus type 5 early region 1 proteins in infected and transformed cells.

Early region 1A (E1A) of human adenovirus type 5 (Ad5) produces two mRNAs coding for phosphoproteins of 289 and 243 residues (289R and 243R). Each of these products has been shown to migrate on sodium dodecyl sulfate gels as two major and two minor species. In the present study, the stabilities of E1A polypeptides, as well as those of some other early Ad5 proteins, were studied in infected KB cells that were pulse-labeled with [35S]methionine and then chased in the presence or absence of cycloheximide. The E1B 58,000- and 19,000-molecular-weight proteins (58K and 19K proteins; 496R and 176R) as well as the E2A 72K DNA-binding protein were relatively stable over the 4-h chase period; turnover was less than 30%. The E1A species were considerably more unstable, with an overall half-life of about 60 min. Interestingly, it was found that when cycloheximide was present during the chase, E1A proteins were much more stable, and the half-life increased to about 240 min. Analysis of the stabilities of individual E1A species indicated that the products of the 1.1-kilobase mRNA (289R) had half lives (about 55 min) somewhat shorter than those (about 90 min) of the 0.9-kilobase mRNA products (243R). In addition, the faster-migrating species produced from each mRNA (molecular weights, 48,500 and 45,000) had significantly shorter half-lives than did the slower-migrating species (52,000 and 50,000). In the presence of cycloheximide, the faster-migrating species were still quite short-lived, but the half-lives of the 52K and 50K species were considerably increased. An examination of the kinetics of turnover of the various E1A species suggested that the faster-migrating forms may be precursors to the slower-migrating ones. Somewhat similar stabilities were also found for the various E1A species in Ad5-transformed 293 cells.     

Complexes of nuclear matrix DNA with proteins tightly bound to DNA contain a specific small-size RNA of a novel type

Analysis of DNA-protein structures composed of nuclear matrix attached DNA and the most tightly bound proteins was performed. Although the previously described non-histone proteins (1) were present the buoyant density of the complex was the same as that of pure DNA. RNA inaccessible to RNase in 0.4 M NaCl but digestible in low ionic strength buffer was detected. This RNA is not a nascent one. It turned out to be homogeneous and represent a novel type of small nuclear RNA. Partial sequence of this RNA is presented.     

Specificity and mode of cleavage of the pH 4.0 endonuclease from adenovirus type 2 - infected KB cells.

Adenovirus type 2 or lambda DNA was digested with the pH 4.0 endonuclease, purified from adenovirus 2-infected KB cells. The enzyme produces a limit digest of approximate size in the range of 140-210 base pairs long. The termini of the DNA fragments generated by the endonuclease digestion had 3'-P and 5'-OH groups. The 3' and 5' end groups of the products were analyzed. Our data indicate that 3' end group was a purine (68-76%), dA occuring about twice the frequency of dG. The 5' end group was either dG or dC with equal frequency. Data obtained by treatment of the 5' labeled endonuclease product of lambda DNA with single-strand specific S1 nuclease from Asperigillus oryzae or exonuclease VII from Escherichia coli indicated that the majority of the products had a short 5' protruding ends. The mode of cleavage of this endonuclease seems to be through initial formation of several single-strand breaks with some base specificity. If these breaks are at close proximity on opposite strands, double-stranded fragments with protruding ends are generated.     

Productive replication of human adenovirus type 5 in canine cells

Development of immunocompetent patient-like models that allow direct analysis of human adenovirus-based conditionally replicative adenoviruses (CRAds) would be beneficial for the advancement of these oncolytic agents. To this end, we explored the possibility of cross-species replication of human adenovirus type 5 (Ad5) in canine cells. With a panel of canine tumor cell lines of both epithelial and mesenchymal derivations, we demonstrate that human Ad5 can productively infect canine cells. Since the biological behavior and clinical presentation of certain dog tumors closely resemble those of their human counterparts, our results raise the possibility of exploiting canine models for preclinical analysis of candidate CRAd agents designed for human virotherapy.     

Adenovirus transcription. IV. Synthesis of viral-specific RNA in human cells infected with temperature-sensitive mutants of adenovirus 5.

Cytoplasmic RNA sequences produced in HeLa cells infected with the adeno-virus 5 temperature-sensitive mutants ts1, ts2, ts9, ts17, ts18, ts19, ts20, ts22, ts49, ts36, and ts125 were characterized by hybridization to DNA probes generated by strand separation of restriction endonuclease fragments of adenovirus 5 DNA. Two "early' mutants defective in DNA synthesis, ts125 and ts36, fail to make wild-type levels of all previously reported classes of late RNA at the nonpermissive temperature. At 40.5 degrees C, both ts125 and ts36 synthesize a wild-type complement of early cytoplasmic RNA 16 h after infection. Under these conditions, no "late' cytoplasmic RNA sequences were observed. Similarly, nuclear RNA present in these cells resembled early cytoplasmic RNA rather than late nuclear RNA. All the late adenovirus 5 temperature-sensitive mutants synthesized normal wild-type levels of late cytoplasmic RNA at the nonpermissive temperature, except ts2, which appears to overproduce certain cytoplasmic species.     

Herpesvirus infection modifies adenovirus RNA metabolism in adenovirus type 5-transformed cells.

The effect of herpes simplex virus (HSV) infection of mRNA metabolism was examined in a system where the fate of specific RNA sequence can be assayed. Adenovirus type 5-transformed rat embryo cell line 107 synthesizes adenovirus-specific RNA (ad-RNA), which functions in the cytoplasm as mRNA. We have utilized ad-RNA as a model for mRNA metabolism, and in a preliminiary study we characterized ad-RNA in the nucleus and cytoplasm by hybridization to filter-bound adenovirus DNA. The results indicated the as-RNA accumulates in the nucleus and that cytoplasmic polyadenylic acid [poly(A)]-containing ad-RNA turns over with a half-life of a few hours. Pulse-chase experiments confirmed these observations and a half-life of about h was determined for the poly(A)-containing cytoplasmic ad-RNA. A second class of ad-RNA remains in the nucleus, where it turns over with a longer hlaf-life (about 24 h). The infection of 107 cells by HSV was restricted at 37 degree C, giving a burst size of 5 PFU per cell and allowing continued host DNA synthesis. Protein synthesis was inhibited greater than 50% by 7 h after infection, and total RNA synthesis was 50% inhibited by 4 h after infection. During the first 8 h after infection, HSV has little effect on the rate of synthesis of ad-RNA as determined by hybridization of nuclear RNA samples, but,during the same period, HSV inhibits the accumulation of poly(A)-containing ad-RNA in the cytoplasm. The degree of this inhibition increases steadily throughout this period and reaches 60% by 6.5 to 8 h after infection. Nosignificant effect was seen on the accumulation of total cellular poly(A)-containing RNA. It was concluded from these experiments that HSV infection alters the metabolism of ad-RNA so as to prevent the normal appearance of the poly(A)-containing mRNA in the cytoplasm. The result for ad-RNA may not represent the behavior of total cellular poly(A)-containing RNA under conditions where infection is restricted.     

Cycl AMP in Gl-arrested BHK21 cells infected with adenovirus type 12

Changes in intracellular levels of cyclic AMP and activity of adenyl cyclase and cyclic AMP phosphodiesterase were studied in G1-arrested BHK21 cells infected with adenovirus type 12 (Ad12). The intracellular concentration of cyclic AMP is reduced at 8 hours after infection (h.p.i.) and reaches a minimum at 14 h.p.i. The decrease in activity of adenyl cyclase is detected at 7.5 h.p.i.; at 9 h.p.i. the activity is about 50% of that in mock-infected cells. No significant changes were observed in the activity of cyclic AMP phosphodiesterase until 13 h.p.i., thereafter the activity of cyclic AMP phosphodiesterase decreased. It is suggested that the observed changes in cyclic AMP metabolism are related to expression of the viral genome, as the first changes are detected only after appearance of Ad12 mRNA in the infected cells.     

Oligo-2',5'-adenylate synthetase activity in cells persistently infected with human T-lymphotropic virus type I (HTLV-I).

Spontaneous production of interferon-gamma (IFN-gamma) was shown in several T-lymphoblastoid cell lines persistently infected with human T-lymphotropic virus (HTLV-1). However, the produced IFN-gamma was not always associated with the induction of the antivirus state. The induction of oligo-2',5'-adenylate synthetase (2-5AS) by IFN was studied in five human T-cell lines persistently infected with HTLV-I (MT-1, MT-2, SMT-1, HUT 102 and OKM-2). Four cell lines are able to produce IFN-gamma spontaneously, while the OKM-2 cell line is not. Poor induction of 2-5AS was recognized in three (MT-1, MT-2 and SMT-1) of the four cell lines producing IFN-gamma, though the poor induction was improved after long-term cultivation of cells with IFN-alpha. On the contrary, in the OKM-2 cell line, significant activity of the enzyme was induced by IFN-alpha. Induction of 2-5AS was not correlated with cell growth inhibition, but with the antivirus state. Furthermore, an inverse relationship between IFN-gamma production and 2-5AS induction was demonstrated in these cell lines with the exception of HUT 102 cells.     

Expression of adenovirus type 2 DNA polymerase in insect cells infected with a recombinant baculovirus.

Sequences encoding adenovirus type 2 DNA polymerase were placed under control of the polyhedrin promoter and inserted into the baculovirus Autographa californica nuclear polyhedrosis virus by homologous recombination. Insect cells infected with the recombinant virus produced substantial amounts of the adenovirus type 2 DNA polymerase protein which was functional in both DNA polymerase and replication initiation reactions. Thus, the baculovirus expression system can provide active adenovirus type 2 DNA polymerase that is produced in quantities suitable for biochemical and structural analysis.