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I’m trying to understand how TMV is expressed and have read (here) that there is a large and small form of the RNA-dependent RNA replicase. These are translated from the same region of the genome, the larger arising from read-through of a leaky stop codon.
Why are there two forms of the replicase? My first impression was that each might produce a sub-genomic RNA, one for the movement protein and one for the capsid protein, but can find no reports confirming this.
Short Answer
As of 2021 the rationale for the production of two proteins from the Tobacco Mosaic Virus (TMV) replicase gene is incompletely understood. The two proteins share some activities but not others, and although they appear to co-operate in the replication process there is a ten-fold excess of the smaller relative to the larger.
Longer Answer
“Why is there a 126-kDa and 183-kDa form of the TMV RNA-dependent RNA replicase?” My answer is based mainly on the review by Buck (Phil. Trans. R. Soc. Lond. B (1999) 354, 613–627), but includes material from some later sources (e.g. Malpica-López et al. (2018)). Many relatively recent papers related to the replicase (e.g. this from 2008, and this from 2012 tend to avoid this question, confining themselves to describing the properties of the two proteins. The main points are:
The only suggestion that Buck makes is that as helicase activity is required for two separate purposes — to unwind double-stranded RNA during replication, and to remove secondary structure from single-stranded RNA when this is used as a template for multiple copies of the complementary strand — the two forms of the replicase may have a different substrate specificity in their helicase activity.
Own thoughts
It is commonplace that the constraints on eukaryotic mRNA translational initiation and the small size of viral genomes have resulted in viruses adopting different strategies for maximizing their coding potential. Read-through is not unique to this class of viruses. I imagine that the replicase preceded the readthrough, in which case one wonders whether the original enzyme was a homo-dimer, which has been superseded by a hetero-dimer with similar protein–protein interaction. The problem here is that the contemporary enzyme has only one active site. Clearly, structural information is needed to address these possibilities.
Footnote: Nomenclature
The original question and some sources (mainly in structural biology) refer to the 126-kDa and 183-kDa forms as subunits of the replicase. Most virological sources do not employ this description, which I think is misleading because it ignores 90% of 126-kDa protein which cannot be involved in replication and therefore does not function as a replicase subunit. It also suggests a level of structural analysis that does not exist. Although replicase enzymes often have several subunits, they are generally structurally distinct. The situation with TMV is different.
The RNA-dependent RNA polymerase of the Tobamoviruses exists as a heterodimer expressed by read-through of the RdRP portion of the genome (open reading frames 1 and 2). The products of these ORFs are two proteins of 183 kDa (large replicase subunit) and 126 kDa (small replicase subunit), with the 126 kDa being produced approximately 10x more than the 183 kDa form. The functions and sizes of the various proteins are described well in Watanabe et al., (1999):
The genome of tobacco mosaic virus (TMV) consists of a single-stranded RNA molecule of about 6,400 nucleotides in length with positive polarity, which encodes at least four polypeptides: 126- and 183-kDa proteins required for transcription and replication (hereafter referred to as the 126K and 183K proteins, respectively), a 30-kDa (30K) protein for cell-to-cell virus movement in infected plants, and an 18-kDa protein for virus coat formation. The sequence of the 126K protein is encoded by the 5′-proximal region of the viral genome and includes the methyltransferase and RNA helicase motifs, while the 183K protein is a read-through protein of the 126K open reading frame (ORF) and contains, in addition to the above two motifs, the RNA-dependent RNA polymerase motif. The RNA polymerase is considered to be involved in both transcription and replication (8). From sequence analysis, it is believed that the viral RNA polymerase contains the 183K protein as a catalytic subunit, but the precise molecular compositions of transcriptase and replicase have not yet been determined.
A common feature of RdRPs in RNA viruses is that they exist as heteromers. A very well known example of this being the RdRP of influenza virus (-ssRNA), which exists as a heterotrimer consisting of the PB1, PB2 and PA subunits produced from the eponymous genome fragments. However, (many) other examples exist in the virus world, including in another plant virus genus Potyvirus, which, in common with Tobamoviruses and 90% of plant viruses, is a positive sense single-stranded RNA virus. The features of the Potyvirus RdRP have just been published, and to quote the linked article (and references therein):
The RdRp–RdRp self-interaction seems to be a common feature for positive-sense, single-stranded RNA viruses, including insect-, animal- and human-, and plant-infecting viruses [38,49,50,51,52,53]. The dimerization or oligomerization of RdRps may increase the stability of these enzymes and protect against degradation.
As you can see from the information provided in the top quote, the functions of the different subunits of the protein are different but complementary. The 126 kDa contains a helicase and methyltransferase motifs, while the 183 kDa functions as the polymerase as well as containing the same helicase and methyltransferase motifs. Lewandowski and Dawson (2000) found that the 183 kDa subunit was capable of performing all the functions listed above, acting as the full RdRP, but with the 126 kDa subunit those functions were performed ~10 times faster. They also found that mutating a base in the helicase domain of the 126 kDa (183 kDa supplied by a helper virus that only expresses the 183 kDa form) resulted in no replication of the RNA with the mutant 126 kDa indicating that this protein is essential to RNA replication. It should also be noted here as per comments, that the evidence for heterodimerism of the two forms is not in the form of a crystal structure, but in a 1:1 stoichiometry in immunoprecipitation as discussed in the Watanabe paper linked above. To my knowledge no-one has produced a full crystal structure of the RdRP of any Tobamovirus.
However, as you might have noticed when you looked at the information on the link you provided, that there seemed to be only about 4 ORFs, and a small number of proteins produced, and you might be thinking something along the lines of
"how does a virus manage to work when it only produces so few proteins?"
The answer to which is viral proteins perform many functions (this feature isn't specific to viral proteins). In particular, the small subunit (126 kDa) seems to act as a suppressor of the host silencing RNA system (siRNA). siRNA systems in plants function a bit like an immune system response - they signal within the cell and extra-cellularly to induce a viral RNA degrading response, so that the virus can not easily spread or establish itself in the host. I would speculate that what better place to suppress siRNA functions than at the site of viral RNA replication itself.
