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Published on: December 9, 2022
Mutational analysis of the SDD sequence motif of a PRRSV RNA-dependent RNA polymerase
Yan Zhou1, Haihong Zheng, Fei Gao
1Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
Abstract:
The subgenomic mRNA transcription and genomic replication of the porcine reproductive and respiratory syndrome virus (PRRSV) are directed by the viral replicase. The replicase is expressed in the form of two polyproteins and is subsequently processed into smaller nonstructural proteins (nsps). nsp9, containing the viral replicase, has characteristic sequence motifs conserved among the RNA-dependent RNA polymerases (RdRp) of positive-strand (PS) RNA viruses. To test whether the conserved SDD motif can tolerate other conserved motifs of RNA viruses and the influence of every residue on RdRp catalytic activity, many amino acids substitutions were introduced into it. Only one nsp9 substitution, of serine by glycine (S3050G), could rescue mutant viruses. The rescued virus was genetically stable. Alteration of either aspartate residue was not tolerated, destroyed the polymerase activity, and abolished virus transcription, but did not eliminate virus replication. We also found that the SDD motif was essentially invariant for the signature sequence of PRRSV RdRp. It could not accommodate other conserved motifs found in other RNA viral polymerases, except the GDD motif, which is conserved in all the other PS RNA viruses. These findings indicated that nidoviruses are evolutionarily related to other PS RNA viruses. Our studies support the idea that the two aspartate residues of the SDD motif are critical and essential for PRRSV transcription and represent a sequence variant of the GDD motif in PS RNA viruses.
Insights
The porcine reproductive and respiratory syndrome virus (PRRSV) replicase nsp9
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- Porcine reproductive and respiratory syndrome virus (PRRSV) relies on its viral replicase for transcription and replication.
- The PRRSV replicase, specifically nsp9, contains conserved motifs characteristic of RNA-dependent RNA polymerases (RdRp) in positive-strand (PS) RNA viruses.
- The SDD motif within nsp9 is crucial for PRRSV's enzymatic functions.
Purpose of the Study:
- To investigate the functional tolerance of the conserved SDD motif in PRRSV nsp9 to amino acid substitutions.
- To determine the impact of individual residues within the SDD motif on RdRp catalytic activity, transcription, and replication.
- To explore the evolutionary relationship between PRRSV and other PS RNA viruses by analyzing conserved motifs.
Main Methods:
- Site-directed mutagenesis was employed to introduce various amino acid substitutions into the SDD motif of PRRSV nsp9.
- Mutant viruses were generated and assessed for genetic stability, polymerase activity, transcription, and replication.
- Sequence analysis was performed to compare the PRRSV SDD motif with conserved motifs in other RNA viral polymerases.
Main Results:
- Only a single substitution, serine to glycine at position 3050 (S3050G), rescued mutant viruses, yielding a genetically stable virus.
- Alterations to the aspartate residues within the SDD motif abolished polymerase activity and viral transcription, but not replication.
- The SDD motif proved invariant and could not accommodate other conserved RNA viral polymerase motifs, except for the GDD motif found in other PS RNA viruses.
Conclusions:
- The two aspartate residues in the PRRSV nsp9 SDD motif are essential for viral transcription.
- The SDD motif represents a sequence variant of the GDD motif, underscoring the evolutionary relatedness of nidoviruses to other PS RNA viruses.
- These findings provide critical insights into the functional constraints and evolutionary history of viral RNA polymerases.
