Cis- and trans-acting elements in cowpea mosaic virus RNA replication

Area of Science:

• Plant virology
• Molecular biology
• RNA replication mechanisms

Background:

• Cowpea mosaic virus (CPMV) has two RNA components: B-RNA for replication and M-RNA for capsid proteins and movement.
• B-RNA self-replicates, while M-RNA requires B-RNA for replication.
• Understanding the cis/trans requirements for CPMV RNA replication is crucial for viral biology.

Purpose of the Study:

• To investigate the replication requirements of CPMV B-RNA and M-RNA.
• To determine the role of non-coding regions and specific protein domains in RNA replication.
• To elucidate the cis- and trans-acting mechanisms governing CPMV RNA replication.

Main Methods:

• Construction and replication analysis of heterologous sequence insertion mutants in CPMV B-RNA.
• Co-inoculation experiments with wild-type and mutant CPMV RNAs in protoplasts.
• Analysis of M-RNA replication using B-RNA as a helper, focusing on non-coding regions and protein domains.

Main Results:

• Mutant B-RNAs with insertions in the coding region failed to replicate, even in the presence of helper B-RNA, demonstrating B-RNA replication is not trans-acting.
• M-RNA replication occurred in trans, dependent on B-RNA encoded proteins.
• Mutant M-RNA transcripts, even with B-RNA non-coding regions, replicated efficiently with B-RNA, suggesting non-coding regions are not primary determinants of cis/trans replication.
• Replication of M-RNA required the N-terminal domain of its encoded 58K protein.

Conclusions:

• CPMV B-RNA replication is a cis-acting process and cannot be complemented in trans.
• CPMV M-RNA replication is trans-acting, requiring factors encoded by B-RNA.
• The N-terminal domain of the M-RNA 58K protein is essential for M-RNA replication.
• Replication of CPMV RNAs is not primarily dictated by their non-coding regions.