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Completion of avian retroviral DNA replication intermediates inhibited by antisense RNA
1Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington.
Abstract:
High level expression of RNA complementary to either neo(r) or src sequences located near the 3' end of recombinant retroviral vectors derived from Rous sarcoma virus inhibited viral replication. Stable integration of proviral DNA was not detected in the presence of antisense RNA. We investigated the mechanism of this inhibition by determining the structure of unintegrated viral DNA (vDNA) intermediates accumulating in the presence of the anti-sense RNA. The major vDNA intermediate detected was a full-length duplex linear molecule with complementary single-stranded long terminal repeats (LTRs). These vDNA linears could be joined directly by T4 DNA ligase to form junctions which contained a single normal LTR. These results can be explained by arrest of linear vDNA formation before strand displacement results in completion of the LTRs. Isolation of these sticky-ended intermediates as linear rather than nicked circular molecules suggests that these complementary vDNA LTR segments were not hydrogen bonded in the infected cell and further implies that completion of LTR synthesis is an ordered, controlled process.
Insights
Antisense RNA targeting Rous sarcoma virus sequences inhibits viral replication by preventing proviral DNA integration. This occurs due to the accumulation of specific linear viral DNA intermediates, suggesting a controlled LTR synthesis process.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Antisense RNA can inhibit gene expression by targeting complementary RNA sequences.
- Rous sarcoma virus (RSV) is a retrovirus used in molecular biology research.
Purpose of the Study:
- To investigate the mechanism by which antisense RNA inhibits replication of recombinant Rous sarcoma virus vectors.
- To determine the structure of viral DNA intermediates formed in the presence of antisense RNA.
Main Methods:
- Expression of antisense RNA targeting neo(r) or src sequences in RSV vectors.
- Analysis of unintegrated viral DNA (vDNA) intermediates using molecular techniques.
- Investigation of vDNA intermediate structure and potential ligation products.
Main Results:
- High-level expression of antisense RNA inhibited viral replication and stable proviral DNA integration.
- Accumulation of full-length duplex linear vDNA intermediates with complementary single-stranded long terminal repeats (LTRs) was observed.
- These linear vDNA molecules could be ligated to form junctions with a single normal LTR, suggesting an arrest in linear vDNA formation.
Conclusions:
- Antisense RNA inhibits RSV replication by interfering with the formation of complete linear viral DNA.
- The accumulation of specific linear vDNA intermediates suggests that the completion of LTR synthesis is a regulated process.