Ribonuclease H activities associated with viral reverse transcriptases are endonucleases.

Area of Science:

• Molecular Biology
• Enzymology
• Virology

Background:

• Reverse transcriptase (RT) enzymes possess ribonuclease H (RNase H) activity, crucial for viral replication.
• The precise mechanism and substrate specificity of viral RNase H activity have been debated, particularly regarding its in vitro versus in vivo behavior.

Purpose of the Study:

• To investigate the exoribonuclease H activity of viral reverse transcriptase.
• To clarify whether viral RTs act as obligatory exonucleases or endonucleases.
• To determine the influence of substrate conformation on viral RNase H activity.

Main Methods:

• Synthesis of novel test substrates, including circular, covalently closed plasmids with ribonucleotide inserts.
• Enzymatic assays using recombinant human immunodeficiency virus RT, avian myeloblastosis virus RT, and Escherichia coli ribonuclease H.
• Degradation assays on capped globin mRNA with a 3' oligo(dT) tail.

Main Results:

• Viral reverse transcriptases (HIV and AMV) cleaved circular, ribonucleotide-containing plasmids, similar to endonuclease activity.
• Avian myeloblastosis virus RT also deadenylated a modified globin mRNA substrate.
• Viral enzymes demonstrated endonuclease-like activity on complex DNA:RNA hybrids, resolving previous controversies regarding their in vitro exonuclease-only function.

Conclusions:

• Viral reverse transcriptases function as endonucleases, not solely exonucleases.
• Viral RNase H activity is highly sensitive to substrate conformation and heteroduplex structure.
• Distinct biochemical properties, including differential inhibition by vanadyl complexes, differentiate viral from cellular ribonuclease H.