Selection of functional 5' cis-acting elements promoting efficient sindbis virus genome replication.

Area of Science:

• Virology
• Molecular Biology
• Genetics

Background:

• The 5' portion of Sindbis virus (SIN) genome RNA is essential for translation and RNA synthesis.
• RNA elements within the first 200 bases of SIN genome RNA play critical roles in both negative- and positive-strand RNA synthesis.

Purpose of the Study:

• To dissect the functions of the SIN 5' RNA sequence using genetic and biochemical approaches.
• To investigate the impact of replacing SIN 5' sequences with those from Semliki Forest virus (SFV).

Main Methods:

• Genetic manipulation: Replacement of SIN 5' ends in defective-interfering (DI) and genome RNAs with SFV sequences.
• Isolation and characterization of pseudorevertants and evolved viral populations.
• Biochemical analysis of RNA synthesis and replication efficiency.

Main Results:

• Chimeric viruses with replaced 5' ends were initially nonviable, but addition of SIN 5' nucleotides restored negative-strand synthesis.
• Evolved 5'-terminal sequences, characterized by AU-rich elements and oligo(A) stretches, demonstrated high efficiency for negative-strand synthesis and replication.
• Increased AU repeat units correlated positively with replication efficiency.
• Certain 5' modifications restored high replication in BHK-21 cells but impaired it in mosquito cells, suggesting altered cell tropism.

Conclusions:

• Identified specific sequence determinants within the SIN 5' end crucial for efficient RNA replication.
• Demonstrated that evolved 5' sequences can enhance viral replication and influence cell tropism.
• Suggests potential strategies for developing alphavirus vectors with restricted cell tropism and optimized packaging.