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.

DNA synthesis primed by mononucleotides (de novo synthesis) catalyzed by HIV-1 reverse transcriptase: tRNA(Lys,3) activation.

HIV-1 RT is able to catalyze DNA synthesis starting from mononucleotides used both as minimal primers and as nucleotide substrates (de novo synthesis) in the presence of a complementary template. The rate of this process is rather slow when compared to the polymerization primed by an oligonucleotide. The addition of tRNA(Lys,3) to this system increased the de novo synthesis rate by 2-fold. Addition of low concentrations of agents able to modify protein conformation, such as urea, dimethylsulfoxide and Triton X-100, can activate the de novo synthesis by a factor 2 to 5. A dramatic synergy is observed in the presence of the three compounds since the stimulating effect of tRNA increases 10-15 times. These results suggest that compounds activating RT are able to induce a conformational change of the enzyme which results in a higher specific activity. Primer tRNA seems to play an important role in HIV-1 RT modification(s) leading to a polymerase having a higher affinity for the primer or the dTTP, but not for the template. The specificity of RT for the template is not influenced by changes in the kinetics or in the thermodynamic parameters of the polymerization reaction.

The ribonuclease H activity of HIV-1 reverse transcriptase: further biochemical characterization and search of inhibitors.

A recombinant homodimer p66/p66 of the HIV-1 reverse transcriptase (RT) was expressed in and purified from a protease-deficient strain of the yeast Saccharomyces cerevisiae. The RNase H activity associated with the homodimer was biochemically characterized. The effect of cations and the hybrid substrate specificity were studied. Some compounds which have been found to inhibit retroviral replication were tested as potential inhibitors of the retroviral DNA polymerase and RNase H activities. Most of these compounds inhibited preferentially the DNA polymerase activity. On the other hand, only suramin was found to inhibit RNase H more efficiently than DNA polymerase. As in the case of the DNA polymerase activity, the thiol-reacting agent N-ethylmaleimide (NEM) did not affect the RNAse H activity of HIV RT. When the effect of NEM was tested against E coli RNase H, a weak inhibitory effect was detected. Surprisingly, NEM strongly inhibits the same bacterial RNase H in the presence of a recombinant form of HIV RT devoid of nuclease activity. These results strongly suggest an interaction between E coli RNase H and HIV-1 RT.