Viral RdRp and its sequence analysis challenges

Viral RdRp protein

Viral RNA dependent RNA polymerase (RdRp) is the protein responsible for copying the genome of an RNA virus. Almost all RNA viruses make an RdRp protein (Wikipedia article on RdRp). Exceptions include RNA retroviruses, where the polymerase protein is a reverse transcriptase, and satellite viruses such as Hepatitis Delta Virus which require RdRp from another virus (the so-called helper virus) to be present to support replication.

RdRp proteins are not named RdRp

Typically, the protein with RdRp function is not officially called RdRp. For example, in nidoviruses the RdRp protein is called Nsp9 or Nsp12, and in the lambda 3 phage it is simply called the lambda protein (see PDB 1MUK). This complicates database searches by gene name.

Where does RdRp sequence terminate?

Sometimes, RdRp has a simple coding sequence (CDS) beginning with a START codon and ending at a STOP codon. In such cases, it is straightforward to identify RdRp as the nucleotide or translated amino acid sequence of this CDS (assuming of course that you know the genetic code of the host). However, things are not always that simple. RdRp often occurs in a longer ORF which codes for multiple proteins; for example in SARS-CoV-2 RdRp function is found in Nsp12, one of several non-structural proteins coded in the long ORF1ab (21,555nt) which accounts for most of the genome (29,903nt). ORF1ab is translated into a long peptide (pp1ab), which is then split into mature proteins by a cleavage enzyme.

In at least one family (narnaviruses), RdRp is not a single protein; it is assembled as a complex from preptides coded in multiple genome segments (https://pubmed.ncbi.nlm.nih.gov/34688782/).

Cleavage sites are hard to find

Unlike START and STOP codons, cleavage sites are not easily recognized by sequence analysis, and in fact are not known in many fully-sequenced genomes (https://pubmed.ncbi.nlm.nih.gov/27567259/. Therefore, the beginning and end of the RdRp sequence in a genome or metagenomic contig may be difficult to identify. In practice, this problem means that predicted RdRp sequences are often truncated or trimmed to shorter subsequences such as a partial domain.

RdRp domains

The RdRp protein is usually constructed from two or more domains. The term domain generally means a segment which folds independently and may be found in combination with other domains in different proteins, but the definition is not precise and may be used in different ways in different contexts.

Domain boundaries are fuzzy

It can be clear that some residues are in a particular domain (for example, the [G/S]DD motif is the palm domain). However, there are no sharp boundaries where two adjacent residues are definitively in different domains. Thus, in contrast to CDS which has clear boundaries (start / stop codons), domain boundaries are fuzzy.

Palm domain

The palm domain is found in all known viral RdRps (Jia and Gong 2019, te Velthuis 2014). Other named domains are usually present in an RdRp protein; for example, nidoviruses such as SARS-CoV-2 have a NiRAN domain at the N terminal before the palm, and reoviruses have an unnamed N-terminal domain before the palm and a bracelet domain after the palm. Regions outside of the palm domain are generally not well understood, and it is often not known whether they are essential for RdRp function or perform some other function.

Varying domains in RdRp

As the examples of nidoviruses (NiRAN+palm) and reoviruses (N-terminal+palm+bracelet) illustrate, the domain content of RdRp varies in different families, and except for the palm domain are often not well characterised. This adds to the difficulty of identifying the boundaries of the RdRp coding sequence in genomes which are far diverged from well-characeterized viruses, and similarly in metagenomic contigs.

Permuted domains

Several families have permuted RdRp genes (Sabanadzovic2009, Ambrose2009, Ferrero2021, Gorbalenya2002) where the palm domain is permuted.