Selection for gene junction sequences important for VSV transcription.

Area of Science:

• Virology
• Molecular Biology
• Gene Regulation

Background:

• Vesicular stomatitis virus (VSV) gene expression relies on intergenic regions (IGRs) and heptauridine (U) tracts for mRNA synthesis and termination.
• The N/P gene junction is critical for maintaining optimal molar ratios of N and P proteins essential for viral RNA replication.

Purpose of the Study:

• To investigate the independent roles of the U tract and IGR in downstream gene transcription.
• To determine how alterations in the N/P gene junction affect viral replication and protein ratios.

Main Methods:

• Engineered VSV with modified N/P gene junction IGRs and U tracts.
• Passaged engineered viruses in cell culture to apply selective pressure for replication.
• Monitored viral replicative ability and sequenced altered intergenic junctions.

Main Results:

• Shortened U tracts reduced viral replication, while mutations restoring U tract length increased it.
• Extended IGRs decreased downstream transcription but had less impact than shortened U tracts.
• Increased P mRNA and protein abundance improved N:P protein molar ratios in selected variants.

Conclusions:

• The length of the upstream gene end U tract is a key factor in efficient downstream gene transcription in VSV.
• Viral evolution can select for mutations that restore U tract length, enhancing viral fitness.
• While IGRs influence transcription, U tract size plays a more critical role in regulating downstream gene expression.