Characterization of the cDNA synthesized by avian retrovirus reverse transcriptase using 35 S avian myeloblastosis virus RNA and an exogenous bovine primer tRNA.

Bovine tRNA(Trp) can be partially hybridized to the avian myeloblastosis virus (AMV) 35 S RNA at 37 degrees C, in the presence of AMV RNA-dependent DNA polymerase (reverse transcriptase). This template-primer complex is active in the synthesis of viral cDNA. The size of the cDNA products synthesized in the in vitro reconstituted AMV system was determined by urea-polyacrylamide gel electrophoresis using a tRNA labelled at the 3'-end by yeast tRNA nucleotidyl transferase. The synthesized cDNA has a size of about 100 nucleotides and was shown by Southern blotting to be complementary to a specific sequence of the 5'-end of the retroviral genome. These results indicate that reverse transcriptase is able to anneal the exogenous primer tRNA at the 'primer-binding site' near the 5'-end of the long terminal repeat (LTR) of AMV RNA.

Involvement of tRNA in retrovirus expression: biological implications of reverse transcriptase-primer tRNA interactions.

We have previously studied the topographical and functional implications of the recognition of primer tRNATrp by avian retrovirus reverse transcriptase. Here we have presented evidence that the enzyme is able to deacylate beef liver Trp-tRNATrp, provided that 35-S viral RNA is present in the incubation mixture. No effect of dNTPs on this activity was observed. The extensive modification of tRNATrp with acrylonitrile led to a marked loss of priming activity by tRNATrp if the annealing between primer and template was performed at 37 degrees C, while the annealing of cyanoethylated tRNA with the viral genome at 75 degrees C gave almost normal levels of cDNA synthesis. We have also studied the priming behaviour of tRNATrp, modified by incorporation of various analogs of adenosine. Only tRNATrp-2'dA was active in cDNA initiation; 3'dA, 3'NH2-3'dA, and primer tRNA with formycin in the 3' end showed low or nonexistent priming activity.

Reverse transcriptase mediated binding of primer tRNA to the viral genome.

A complex between tRNATrp (beef) and 35 S RNA from avian myeloblastosis virus is obtained when the mixture is preincubated in the presence of reverse transcriptase at 35 degrees C. The tRNA-RNA complex is active in initiating DNA synthesis catalyzed by reverse transcriptase. The interaction of tRNA with reverse transcriptase involves the partial unwinding of the acceptor stem of tRNA, as evidenced by nuclease digestion with RNAase T1 and micrococcal nuclease. When tRNA2Glu (coli), having a high degree of similarity with primer tRNA at the level of the acceptor stem, was used as primer for DNA synthesis, a low but significant level of incorporation was obtained, if the reaction was performed at 35 degrees C, while a high incorporation, similar to the one obtained with tRNATrp was obtained when the annealing between tRNA2Glu and 35 S RNA was performed at 80 degrees C. Our evidences point out to an important role of the viral DNA polymerase in positioning the primer on the RNA genome.

Formycin 3' end modified tRNATrp. Recognition by avian myeloblastosis virus reverse transcriptase and primer function.

Primer tRNATrp has been modified at the 3' end by adenosine analogues: 2'deoxyadenosine, 3'deoxyadenosine, 3' amino-3' deoxyadenosine and formycin. Aminoacylation of modified tRNATrp with cognate aminoacyl-tRNA synthetase and primer function for DNA synthesis catalyzed by AMV reverse transcriptase have been studied. The tRNATrp was able to accept tryptophan but did not initiate the DNA synthesis directed by 35S AMV RNA. Recognition of modified tRNATrp by AMV reverse transcriptase was not affected as followed by enzyme-tRNA complex formation. The functional consequences of these effects are discussed.

Small finger protein of avian and murine retroviruses has nucleic acid annealing activity and positions the replication primer tRNA onto genomic RNA.

Retrovirus virions carry a diploid genome associated with a large number of small viral finger protein molecules which are required for encapsidation. Our present results show that finger protein p12 of Rous sarcoma virus (RSV) and p10 of murine leukaemia virus (MuLV) positions replication primer tRNA on the replication initiation site (PBS) at the 5' end of the RNA genome. An RSV mutant with a Val-Pro insertion in the finger motif of p12 is able to partially encapsidate genomic RNA but is not infectious because mutated p12 is incapable of positioning the replication primer, tRNATrp. Since all known replication competent retroviruses, and the plant virus CaMV, code for finger proteins analogous to RSV p12 or MuLV p10, the initial stage of reverse transcription in avian, mammalian and human retroviruses and in CaMV is probably controlled in an analogous way.

Inhibition of Rous sarcoma virus production by formycin.

The effect of formycin, an adenosine analog, on the growth of chick embryo fibroblasts and on Rous sarcoma virus (RSV) production was studied. An adverse effect on cell proliferation was observed in the presence of 10 microM formycin. Treatment with 5 microM formycin for 24 hr reduced by a factor of about 1000 the yield of infections progeny whereas the cell growth remained unaltered. Moreover the few particles released in the presence of formycin showed a markedly decreased ability to synthesize viral cDNA. This impairment was shown to be related to a nonfunctional primer tRNA.

Avian myeloblastosis reverse transcriptase deacylates tryptophanyl-tRNA.

Tryptophanyl-tRNA can be used as a primer for RNA-dependent DNA synthesis by avian reverse transcriptase. We have determined that whereas the retroviral polymerase is not by itself capable of deacylating Trp-tRNATrp, the ester linkage between the 3' OH of the ribose moiety and the aminoacid can be very efficiently hydrolyzed when both the polymerase and the viral 35 S RNA are present.

The in vitro inhibition of DNA polymerase alpha and avian reverse transcriptase by novobiocin.

Novobiocin inhibits animal DNA polymerase alpha and avian reverse transcriptase activities when these enzymes are assayed in vitro with activated DNA as template. Under the same conditions DNA polymerase beta and gamma are much less inhibited. DNA polymerase alpha and reverse transcriptase are inhibited by different mechanisms: in the case of the retroviral enzyme the effect of novobiocin is not overcome by dilution of the drug, while in the case of polymerase alpha the inhibition disappeared after novobiocin dilution. The inhibition of polymerase alpha by novobiocin is non-competitive with respect to the TTP precursor or activated DNA. The irreversible inactivation of reverse transcriptase by novobiocin leads to the loss of the enzyme affinity for primer tRNATrp. Moreover, novobiocin inhibits the partial unwinding of the 3' end of tRNATrp by reverse transcriptase.