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Richard joins TWiV to discuss the SARS-CoV-2 antiviral drug Molnupiravir , including how it was discovered, its mechanism of action, whether it is a mutagen for cells, and the future of drugs for treatment of COVID-19.
Hosts: Vincent Racaniello, Alan Dove, and Brianne Barker
Guest: Richard Plemper
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RNA viruses exist close to their error threshold, the point beyond which additional mutations cause loss of infectivity. It has been suggested that RNA recombination prevents viruses from exceeding the error threshold – a situation called error catastrophe – but there has been little experimental support for this hypothesis. An analysis of poliovirus RNA synthesis suggests that this hypothesis is correct.
[Read more…] about Avoiding error catastrophe with recombination
From ASM Microbe 2019, Vincent, Brianne and Calvin meet up with Craig Cameron to discuss his career and his work exploring RNA-dependent RNA synthesis and single cell virology.
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A new member of the arenavirus family, Lujo virus, has been identified in patients who died during an outbreak of hemorrhagic fever in late 2008. Sequence analysis reveals that Lujo is a new arenavirus, genetically distinct from other members of the family which includes Lassa virus.
A patient with unexplained hemorrhagic fever was identified in Zambia in September 2008. After transfer to Sandton for further care, four health care workers involved with the case also became ill. The first four patients died, and the fifth survived after treatment with ribavirin.
Sequence analysis of total liver and serum RNA from the patients revealed the presence of a new member of the arenavirus family. The virus has been provisionally named Lujo based on its origin in Lusaka, Zambia, and Johannesburg, South Africa. Phylogenetic trees were prepared from the viral genome sequences (example at left). These trees show that Lujo virus branches off the root of the Old World arenaviruses. This analysis suggests that Lujo virus is a member of a novel genetic lineage, and is distinct from previously characterized viruses in this family.
It’s not clear how the index case was infected by Lujo virus. Most arenavirus infections are acquired by contact with infected rodent excreta, by inhalation of dust or aerosolized virus-containing particles, or ingestion of contaminated foods. At least one arenavirus might be transmitted from bats, and this possibility should not be ruled out for Lujo virus. The index patient likely transmitted the infection to the four health care workers via aerosols or infected body fluids.
There are several noteworthy aspects of this emerging story. First, it is clear that pyrosequencing can be used to rapidly identify new pathogens. In this case, sequences were obtained within 72 hours of receipt of clinical specimens. Lujo virus appears to be highly lethal in humans, based on the deaths of 4 out of 5 of the infected patients. However the number of infections identified so far is small, and the lethality ratio might fall as additional infections are studied. Now that the sequence of Lujo virus is known, reagents can be produced which will enable rapid identification of new cases by more conventional diagnostic approaches such as polymerase chain reaction. Lujo is a new member of the arenavirus family and is genetically distinct from both Old and New World arenaviruses. Why it has just appeared in humans is not known, but possible reasons include the more frequent contact between humans and wild animal species, and better and more aggressive detection methods. It is likely that a virus closely related to Lujo is present in either rodents or bats but has not been previously detected. As we have said many times before, the zoonotic pool is very deep.
I am struck by similarities between the events surrounding the discovery of Lujo virua and Lassa virus in 1969. In both outbreaks, Columbia University Medical center was involved, and a nurse who was infected by the index patient survived as a consequence of antiviral therapy.
Briese, T., Paweska, J., McMullan, L., Hutchison, S., Street, C., Palacios, G., Khristova, M., Weyer, J., Swanepoel, R., Egholm, M., Nichol, S., & Lipkin, W. (2009). Genetic Detection and Characterization of Lujo Virus, a New Hemorrhagic Fever–Associated Arenavirus from Southern Africa PLoS Pathogens, 4 (5) DOI: 10.1371/journal.ppat.1000455
The capacity of RNA viruses to produce prodigious numbers of mutations is a powerful advantage. But remember that selection and survival must balance genetic fidelity and mutation rate. Many mutations are not compatible with viral replication. Consequently, if mutation rates are high, at some point accumulating base changes lead to lethal mutagenesis – the population is driven to extinction. The error threshold is a mathematical measurement of the genetic information that must be maintained to ensure survival of the population. The results of experiments clearly show that RNA viruses evolve close to their error threshold.
The first clues that viruses exist on the edge came from studies in which cells infected with the RNA viruses poliovirus and vesicular stomatitis virus were treated with base analogs that act as mutagens. These compounds, such as 5-azacytidine, are incorporated into the growing RNA chain during replication, leading to incorporation of incorrect bases. Treatment of virus infected cells with such drugs dramatically inhibits the production of new infectious virus particles, but stimulates mutagenesis of the viral RNA by only 2- or 3-fold. When the same experiment is done with cells infected with a DNA virus, the mutation frequency increases by several orders of magnitude.
A direct demonstration of error catastrophe was achieved by treating poliovirus-infected cells with the mutagen ribavirin. Treatment with a concentration of ribavirin that causes a 9.7-fold increase in mutagenesis lead to a 99.3% loss in poliovirus infectivity. A graph of the results shows that poliovirus exists at the edge of viability.
The line on the graph shows the infectivity of poliovirus RNA as a percentage of untreated viral RNA. The introduction of 2 mutations per genome reduces infectivity to about 30% of wild type RNA, while infectivity is nearly eliminated with 7 mutations per genome.
Ribavirin pushes poliovirus beyond the error threshold because it causes the RNA polymerase to make more errors than it already does. What would be the property of a poliovirus mutant resistant to this drug? A poliovirus mutant resistant to ribavirin was selected by passing the virus in the presence of increasing concentrations of the drug. Resistance to ribavirin is caused by a single amino acid change, G64S, in the viral RNA-dependent RNA polymerase. RNA synthesis of ribavirin-resistant poliovirus is characterized by fewer errors, in the absence of drug, compared with the parental virus. Therefore the mechanism of resistance to the mutagen ribavirin is achieved through an amino acid changes that makes a viral RNA polymerase with greater fidelity.
Now that we have an RNA polymerase that makes fewer errors, we can use it to limit viral diversity and test the theory that viral populations, not individual mutants, are the target of selection.
Crotty, S. (2001). RNA virus error catastrophe: Direct molecular test by using ribavirin Proceedings of the National Academy of Sciences, 98 (12), 6895-6900 DOI: 10.1073/pnas.111085598
Pfeiffer, J. (2003). A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity Proceedings of the National Academy of Sciences, 100 (12), 7289-7294 DOI: 10.1073/pnas.1232294100