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error rate

TWiV 83: An hour with Dr. Kiki

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Hosts: Vincent Racaniello, Alan Dove, Rich Condit, and Kirsten Sanford

On episode #83 of the podcast This Week in Virology, Vincent, Alan, Rich, and special guest Dr. Kirsten Sanford talk about her career in science media, then consider whether smallpox eradication led to the AIDS pandemic, high fidelity RNA synthesis, and a new Ebola virus vaccine.

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Why don’t DNA based organisms discard error repair?

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quasispecies1The recent series of posts on polymerase error rates and viral evolution has elicited many excellent and thought provoking comments from readers of virology blog. Here is one that I had not thought of before, and which I’ll use on an exam in my virology course:

Here’s a tough question. In the follow up blog to this, you say that the high mutation rates of RNA viruses is beneficial to survival in a complex environment. If this is true, why don’t DNA viruses evolve high mutation rates also? It would be simple for them to delete their proofreading domain.

There is no answer to this question, so I’ll speculate. I believe that DNA viruses have error correction mechanisms so that they can have very long genomes. RNA viral genomes are no longer than 27-31 kb. This limit is probably imposed by their high error rate: if RNA genomes were longer, they would likely sustain too many lethal mutations to survive. Error correction mechanisms allow for DNA viral genomes up to 1.2 million bases in length. Smaller DNA viruses don’t have their own DNA polymerases – they use those of the cell. Cellular DNA polymerases have error repair to avoid mutations that lead to diseases such as cancer. Both forms of reproduction are evolutionarily sustainable: shorter RNAs with lots of errors; longer DNAs with fewer errors.

The reader who posed the original question then came back with this retort:

If reduced fidelity is beneficial to RNA viruses, because of the complex environment they are in, why don’t DNA viruses do the same thing?

I think the same endpoint, in terms of surviving in complex environments, is achieved by both strategies. RNA viruses have high diversity; DNA genomes have many more gene products which allow them to survive in diverse situations. Both strategies appear to be evolutionarily sustainable.

It’s important to keep in mind that the goal of viral evolution is survival. Evolution does not move a viral genome from “simple” to “complex”, or along a trajectory aimed at “perfection”. Change is effected by elimination of the ill adapted of the moment, not on the prospect of building something better for the future.

While researching this subject I came across a series of papers on DNA synthesis by African swine fever virus, a virus with a DNA genome of 168-189 kb. The viral genome encodes a complete DNA replication apparatus, including DNA polymerase and DNA repair enzymes. Incredibly, the DNA repair pathway itself is error-prone, which is believed to contribute to the genetic variability of the virus. There is some controversy concerning the error rate for this virus, so we don’t know the consequence of this observation for fidelity of DNA replication. Nevertheless, these observations suggest that evolution has seen fit to tinker with, and perhaps increase, the error rates of certain DNA based organisms.

Lamarche, B., Kumar, S., & Tsai, M. (2006). ASFV DNA Polymerase X Is Extremely Error-Prone under Diverse Assay Conditions and within Multiple DNA Sequence Contexts. Biochemistry, 45 (49), 14826-14833 DOI: 10.1021/bi0613325

The error-prone ways of RNA synthesis

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Now that we have examined influenza viral RNA synthesis, it’s a good time to step back and look at a very important property of this step in viral replication. All nucleic acid polymerases insert incorrect nucleotides during chain elongation. This misincorporation is one of the major sources of diversity that allows viral evolution to take place at an unprecedented scale. Put another way, viruses are so successful because they make a lot of mistakes.

Nucleic acids are amazing molecules not only because they can encode proteins, but because they can be copied or replicated. Copying is done by nucleic acid polymerases that ‘read’ a strand of DNA or RNA and synthesize the complementary strand. Let’s start by examining DNA synthesis. Below is a DNA chain, which consists of the bases A, G, C or T strung together in a way that codes for a specific protein. In this example, the template strand is at the bottom, and consists of the bases A, C, C, T, G, A, C, G, and G (from left to right). A DNA polymerase is copying this template strand to form a complementary strand. So far the complementary bases T, G, G, A, and C have been added to the growing  DNA chain. The next step is the addition of a T, which is the complementary base for the A on the template strand:

dna-polymerase-1

So far all is well. But all nucleic acid polymerases are imperfect – they make mistakes now and then. This means that they insert the wrong base. In the next step below, the DNA polymerase has inserted an A instead of the correct G:

dna-polymerase-2

Insertion of the wrong base leads to a mutation – a change in the sequence of the DNA. In general, it’s not a good idea to make new DNAs with a lot of mutations, because the encoded protein won’t function well (but there are exceptions, as well will see). But in this case, there is a solution – DNA-dependent DNA polymerases (enzymes that copy DNA templates into DNA) have proofreading abilities. The proofreader is an enzyme called exonuclease, which recognizes the mismatched A-C base pair, and removes the offending A. DNA polymerase then tries again, and this time inserts the correct G:

dna-polymerase-3

Even though DNA polymerases have proofreading abilities, they still make mistakes – on the order of about one misincorporation per 107 to 109 nucleotides polymerized. But the RNA polymerases of RNA viruses are the kings of errors – these enzymes screw up as often as one time for every 1,000 – 100,000 nucleotides polymerized. This high rate of mutation comes from the lack of proofreading ability in RNA polymerases. These enzymes make mistakes, but they can’t correct them. Therefore the mutations remain in the newly synthesized RNA.

Given a typical RNA viral genome of 10,000 bases, a mutation frequency of 1 in 10,000 corresponds to an average of 1 mutation in every replicated genome. If a single cell infected with poliovirus produces 10,000 new virus particles, this error rate means that in theory, about 10,000 new viral mutants have been produced. This enormous mutation rate explains why RNA viruses evolve so readily. For example, it is the driving force behind influenza viral antigenic drift.

Here is a stunning example of the consequences of RNA polymerase error rates. Tens of millions of humans are infected with HIV-1, and every infected person produces billions of viral genomes per day, each with one mutation. Over 1016 genomes are produced daily on the entire planet. As a consequence, thousands of mutants arise by chance every day that are resistant to every combination of antiviral compounds in use or in development.

I cannot overemphasize the importance of error-prone nucleic acid synthesis in RNA viral evolution and disease production. We’ll spend the next two days examining the consequences of error prone replication. First we’ll consider the implications for viruses as a population, and then we’ll discuss the outcome when a virus produces an RNA polymerase that makes fewer mistakes. Please bear with me as we diverge slightly from influenza virus; these concepts will be an important and enduring component of your toolbox of virology knowledge.