Recombinant Simian virus 5 RNA-directed RNA polymerase L (L), partial

What is the structural organization of the SV5 antigenomic promoter recognized by the RNA polymerase L?

The SV5 antigenomic promoter consists of three sequence-dependent elements that are essential for optimal RNA replication:

• Conserved Region I (CRI): A 19-base segment at the 3′ terminus, which is highly conserved among rubulaviruses

• Conserved Region II (CRII): An 18-base internal region located 72-90 bases from the 3′ end, contained within the coding region of the L protein gene

• Third element: Located between bases 51-66 from the 3′ end of the RNA

These elements are separated by sequence-independent spacer regions. Mutational analysis shows that substitution of bases 45-72 with non-viral sequences reduces replication to approximately 3% of wild-type levels, while substitution of bases 21-38 has minimal effect . Proper spacing between these elements is critical, suggesting they must align on the same face of the helical nucleocapsid .

What sequence motifs within CRII are critical for polymerase function?

The essential sequence motifs within CRII consist of two consecutive 5′-CGNNNN-3′ hexamers that form an important sequence in the SV5 CRII promoter element . Experimental evidence shows:

• Replication is significantly reduced by substitutions for two CG dinucleotides, which in the nucleocapsid template are in the first two positions of the first two hexamers of CRII nucleotides

• The G residue in the second position within the three hexamers is strictly conserved among rubulaviruses and between the SV5 leader and trailer regions

• Substitutions for other bases within CRII have minimal effects on RNA synthesis

This pattern of sequence conservation suggests a critical role for these specific nucleotides in polymerase recognition and function.

How does spacing between promoter elements affect SV5 RNA replication?

The spacing between promoter elements plays a crucial role in SV5 RNA replication:

• Six-base insertions or deletions in the RNA segment separating CRI from CRII can dramatically reduce RNA replication

• This spacing requirement is independent of the "rule of six" requirement for genome length

• The spacing sensitivity likely reflects the need for CRI and CRII to align on the same face of the helical nucleocapsid template

• The third promoter element (bases 51-66) can be repositioned relative to CRI and CRII without decreasing replication efficiency

When mutant minigenomes were constructed with bases from the third element moved two, four, or six positions closer to CRII, replication levels were maintained or slightly increased compared to wild-type . This indicates that while proper alignment of CRI and CRII is critical, the position of the third element is more flexible.

What experimental systems are used to study SV5 RNA polymerase function?

Several experimental approaches are employed to investigate SV5 RNA polymerase function:

A typical experimental workflow involves:

• Constructing minigenome analogs containing mutations in promoter elements via PCR

• Transfecting human lung cells (A549) infected with vaccinia virus expressing T7 RNA polymerase

• Co-transfecting plasmids encoding NP, P, and L proteins

• Harvesting total RNA after 36-48 hours and analyzing by Northern blotting

• Quantifying relative replication levels compared to wild-type templates

How does the third promoter element (bases 51-66) contribute to RNA replication?

The third promoter element (bases 51-66) shows unique characteristics compared to CRI and CRII:

• Mutational analysis indicates this element is required for optimal RNA replication, with complete replacement reducing replication to ~3-6% of wild-type levels

• Unlike CRI and CRII, this element shows no apparent sequence identity among Rubulavirus antigenomic promoters

• Replication is affected only when the majority of this sequence is replaced, while smaller substitutions have lesser effects

• This suggests a functionally redundant signal distributed across bases 51-66

• Replication is not affected by changes in the position of this element relative to CRI and CRII or the predicted hexamer phase of NP encapsidation

Experimental data show that when six to nine base substitutions were engineered into this region (mutants 10-12), replication was reduced to ~20% of wild-type levels, indicating that important sequences are distributed throughout this region .

What are the methodological approaches for creating recombinant SV5/PIV5 expression vectors?

PIV5 offers several advantages as an expression vector, including its ability to infect many cell types with little cytopathic effect and its cytoplasmic replication without a DNA phase . Key methodological approaches include:

• Insertion of foreign genes:

  • Foreign genes can be inserted into the PIV5 genome as extra genes between existing viral genes (e.g., between HN and L)

  • The hemagglutinin (HA) gene from influenza A virus can be successfully expressed when inserted into this location

• Single-cycle infectious vectors:

  • F-deleted viruses supported by F-expressing helper cell lines allow for safer vector systems

  • These vectors can efficiently express recombinant proteins in multiple cell lines

• Persistent vs. acute phenotypes:

  • Vectors can be engineered with either persistent or acute/lytic phenotypes based on P protein mutations

  • The amino acid at position 157 in P (serine vs. phenylalanine) determines persistence vs. acute infection

• Reporter gene incorporation:

  • Fluorescent proteins (mCherry, GFP) can be incorporated to monitor infection and expression

  • These reporters allow for easy assessment of viral spread and protein expression kinetics

Researchers have demonstrated production of recombinant proteins like monoclonal antibodies at levels of 20-50 mg/L in Chinese hamster ovary cells grown to a density of ~1×10^6 cells/mL .

What is the molecular mechanism of readthrough transcription in SV5/PIV5?

Readthrough transcription occurs when the viral polymerase ignores gene junction termination signals, producing dicistronic or polycistronic mRNAs . The process involves:

• At gene junctions, the polymerase normally terminates transcription and releases a poly(A)+ mRNA before reinitiating at a downstream start site

• The gene end contains a stretch of uridyl (U) residues that direct polyadenylation through a stuttering mechanism

• For paramyxoviruses like SV5, this typically includes a U7 tract (seven uridyl residues)

• Removing even a single U residue can result in high levels of readthrough products

Experimental approaches to study this phenomenon include:

• Creating mutations in gene junctions (e.g., M-F junction)

• Analyzing transcripts via Northern blot to detect dicistronic mRNAs

• Measuring the impact on virus growth characteristics

Understanding readthrough transcription is important for both basic virology research and vector development, as it affects gene expression ratios.

How does the RNA polymerase of SV5/PIV5 compare to other viral RNA-dependent RNA polymerases?

While SV5/PIV5 RNA polymerase shares common features with other viral RNA-dependent RNA polymerases, there are significant differences:

• Promoter recognition: SV5 requires three discontinuous elements (CRI, CRII, and bases 51-66), while some other viruses may have different arrangements

• Terminal complementarity: For rhabdoviruses like vesicular stomatitis virus (VSV), the level of RNA replication can be influenced by terminal complementarity in the promoter region, whereas this appears less important for SV5

• Subgenomic RNA synthesis: Some RNA viruses like caliciviruses utilize a stem-loop structure in the polymerase coding region for subgenomic RNA synthesis, a mechanism distinct from paramyxoviruses

• Nucleotide binding: Calicivirus RdRps have specific positively charged amino acid residues (e.g., Lys169, Lys183, Arg185) involved in RNA recognition and binding near the active site

• Encapsidation requirements: The "rule of six" affects different paramyxoviruses to varying degrees, with some showing stricter adherence than others

Research methodologies examining these differences typically involve comparative studies using reverse genetics systems, mutational analyses, and structural biology approaches.