Extreme GC3 codon bias in a novel brown seaweed virus results in pseudoambigrammatic characteristics

Although viruses are often studied in relation to disease, they can also offer valuable insights into RNA functionality. Due to their vast range of hosts and high mutation rates, viruses can quickly adapt to changing environments. It is therefore likely that viruses exploit a broad range of RNA functionalities to develop strategies for host infection and propagation. Our aim is to discover RNA viruses with survival strategies that reveal unknown RNA characteristics and functionalities. Here, we report a new, unclassified negative-sense single-stranded RNA virus from the large brown seaweed <i>Saccharina latissima</i> that showed a unique genome characteristic. The novel virus, designated SalaUV-NL1, possesses a bipartite genome. The small segment (2120 nt) encodes a putative 328-aa protein and possibly an additional protein of ≥315 aa. The large segment (7065 nt) encodes a putative RNA-dependent RNA polymerase (RdRp) of 2296 amino acids on the anti-genomic strand, but strikingly contains a similarly sized and largely overlapping open reading frame (reverse ORF; rORF) on the genomic strand that encodes a putative protein of 2317 aa. This raises the possibility that SalaUV-NL1 represents an ambigrammatic virus. However, the RdRp ORF exhibits an extreme GC3 codon bias (98%), far exceeding the typical codon constraints of ambigrammatic viruses that mostly reflect the avoidance of stop codons on the reverse strand. Therefore, the rORF is likely incidental because it is the result of the GC3 codon bias on the RdRp-coding anti-genomic strand. We therefore designate SalaUV-NL1 as pseudoambigrammatic. Several other viruses with a similar pseudoambigrammatic signature, characterized by unusually high GC3 codon bias, were identified in GenBank, most of which lack a discernible rORF. The codon bias of SalaUV-NL1 surpasses even that of its host, <i>S. latissima</i>, although other hosts of pseudoambigrammatic viruses do not display elevated GC3 levels. Comparative analysis of multiple SalaUV-NL1 variants further revealed exceptionally high nucleotide substitution rates, particularly silent A/U → G/C transitions, reaching frequencies of up to 81%. This novel virus, therefore, exemplifies a previously unrecognized survival strategy among RNA viruses, the functional implications of which remain unclear.