A study done in 1985 on a 16th century Italian mummy suggested that the two year old child had smallpox. Recent sequence analysis of tissues from the mummy now reveal the presence of hepatitis B virus, not smallpox virus. The sequence of the viral genome suggests that HBV entered the human population well before 1500.
The high mutation rate of RNA viruses enables them to evolve in the face of different selection pressures,Â such as entering a new host or countering host defenses. It has always been thought that the sources of such mutations are the enzymes that copy viral RNA genomes: they make random errors which they cannot correct. Now it appears that a cell enzyme makes an even greater contribution the mutation rate of an RNA virus.
Deep sequencing was used to determine the mutation rate of HIV-1 in the blood of AIDS patients by searching for premature stop codons in open reading frames of viral RNA. Because stop codons terminate protein synthesis, they do not allow production of infectious viruses. Therefore they can be used to calculate the mutation rate in the absence of selection. The mutation rate calculated in this way, 0.000093 mutations per base per cell, was slightly higher than previously calculated from studies in cell culture.
When HIV-1 infects a cell, the enzyme reverse transcriptase converts its RNA genome to DNA, which then integrates into the host cell genome. Identification of stop codons in integrated viral DNA should provide an even better estimate of the mutation rate of reverse transcriptase, because mutations that block the production of infectious virus have not yet been removed by selection. The mutation rate calculated by this approach was 0.0041 mutations per base per cell, or one mutation every 250 bases. This mutation rate is 44 times higher than the value calculated from viral RNA in patient plasma (illustrated).
Sequencing of integrated viral DNA from many patients revealed that the vast majority of mutations leading to insertion of stop codons – 98% – were the consequence of editing by the cellular enzyme APOBEC3G.Â This enzyme is a deaminase that changes dC to dU inÂ theÂ first strandÂ ofÂ viralÂ DNA synthesizedÂ byÂ reverseÂ transcriptase. APOBEC3G constitutes an intrinsic defense against HIV-1 infection, because extensive mutation of the viral DNA reduces viral infectivity. Indeed, most integrated HIV proviruses are not infectious as a consequence of APOBEC3G-induced mutations. That infection proceeds at all is due to incorporation of the viral protein vif in the virus particles. Vif binds APOBEC3G, leading to its degradation in cells.
The mutation rate of integrated HIV-1 DNA calculated by this method is much higher than that of other RNA viruses. This high mutation rate is driven by the cellular enzyme, APOBEC3G. At least half of the mutations observed in plasma viral RNAs are also contributed by this enzyme.
It has always been thought that error-prone viral RNA polymerases are largely responsible for the high mutation rates of RNA viruses. The results of this study add a new driver ofÂ viral variation, a cellular enzyme. APOBEC enzymes are known to introduce mutations in the genomes of other viruses, including hepatitis B virus, papillomaviruses, and herpesviruses. Furthermore, the cellular adenosine deaminase enzyme can edit the genomes of RNA viruses such as measles virus, parainfluenza virus, and respiratory syncytial virus. Cellular enzymes may therefore play a much greater role in the generation of viral diversity than previously imagined.
Virus populations do not consist of a single member with a defined nucleic acid sequence, but are dynamic distributions of nonidentical but related members called a quasispeciesÂ (illustrated at left). While next-generation sequencing methods have the capability of describing a quasispecies, the errors associated with this technology have limited progress in our understanding of the genetic structure of virus populations. A new methodÂ calledÂ CirSeq reduces next-generation sequencing errors to allow an accurate description of viralÂ quasispecies.
The key to eliminating sequencing errors is a clever approach based on the conversion of viral RNAs to circular molecules. When copied with reverse transcriptase, tandemly repeated cDNAs are produced (illustrated below). Mutations in the original viral RNA will be shared by all repeats derived from a circle, but not errors produced during copying or sequencing. The latter can be computationally subtracted, reducing sequencing error to a point that is much lower than the estimated mutation rate of an RNA virus.
CirSeq was used to characterize poliovirus populations produced by seven serial passages in HeLa cells. The calculated mutation frequency, 2 X 10-4 mutations per nucleotide, was substantially lower compared with estimates determined by conventional sequence analysis. Over 200,000 sequence reads per nucleotide position were used to detect >16,500 variants per population per passage. This numberÂ represents ~74% of all possible alleles. Many mutations were detected at nearly all positions in the viral RNA. Most mutations occur at a frequency between 1 in 1000 to 1 in 100,000. The conclusion is that the virus population produced in HeLa cells consists mainly of genomes with the consensus sequence, and small amounts of many variantÂ genomes. These variants are only those that give rise to viable viruses; lethal mutations are not observed.
CirSeq was also used to calculate the mutation rate of poliovirus. The rates vary according to type: transitions occurred at a rate of 2.5 X 10-5 to 2.6 X 10-4 substitutions per site, while transversions were observed at a rate of 1.2 X 10-6 to 1.5 X 10-5 substitutions per site. Nucleotide-specific differences in mutation rate were also observed: C to U and G to A transitions were 10 times more frequent than U to C and A to G. These rates are consistent with previously determined values using other methods.
This method can also be used to determine the fitness of each base at everyÂ position in the genome, according to changes observed during the seven passages in HeLa cells. This analysis allows determination of which bases are neutral, and which are selected, and when combined with analysis of protein structure, can provide new insights into viral functions.
By enablingÂ a sequencing approach that givesÂ an accurate description of virus populations at a single-nucleotide level,Â CirSeq can be used to provide an unprecedented view of how virus populations change during evolution.
The Food and Drug Administration has approved the use of a single pill containing two different antiviral drugs for theÂ treatment for hepatitis C. It is the first combination pill approved for the disease, and also the first treatment that does not contain interferon or ribavirin.
The new hepatitis C drug, called Harvoni, is a mixture ofÂ the antiviral drugs ledipasvir and sofosbuvir. Ledipasvir (pictured) is an inhibitor of the hepatitis C virus protein NS5A, which has multiple roles in the viral replication cycle that include RNA synthesis and virus particle assembly.Â The mechanism of NS5A inhibition by ledipasvir is not known. Sofosbuvir is a previously licensed inhibitor that targets the viral RNA-dependent RNA polymerase. It is an analog of the nucleoside uridine, one of the four building blocks of RNA. Sofosbuvir is utilized by the viral RNA polymerase, leading to inhibition of viral RNA synthesis.
The use of single antiviral drugs (monotherapy) to treat RNA virus infections is always problematic because resistance usually arises rapidly. Dual-therapy pills like Harvoni are better, but the best are triple-therapy pills. Triple therapy formulationsÂ such as AtriplaÂ have been used successfully to treat infections with HIV-1, and presumably there willÂ be mixtures of three antiviral drugs for treating hepatitis C.
Let’sÂ use HIV-1 to illustrate the value of treating infections with multiple antiviral drugs. The HIV-1 viral genome, like that of HCV, is slightlyÂ less than 10,000 bases long. Assume that one mutation in the viral genome is needed for drug resistance. If the RNA polymerase mutation rate is 1 out of every 10,000 basesÂ synthesized, thenÂ each base in the viral genome is substituted in a collection of 10,000 viruses. An HIV-1 infected person can make as many as 10,000,000,000 virus particles each day, soÂ 1010/104 = one million viruses will be produced each day with resistance to one drug.
If we use two antiviral drugs, developing resistance to both occurs in everyÂ 104 xÂ 104 = 108 viruses. In this caseÂ 1010/108Â = 100Â viruses will be produced each day with resistance to twoÂ drugs.
If we use threeÂ antiviral drugs, developing resistance occurs in everyÂ 104Â xÂ 104Â xÂ 104= 1012Â viruses, which is more than what is produced each day.
This is why triple antiviral therapy has been so successful for the treatment of AIDS.
And yes, I’m sure someone has tested Sofosbuvir for inhibition of Ebola virus replication.
On episode #207 of the science show This Week in Virology,Â Vincent, Alan, Matt, and Kathy review the use of silk to stabilize antibiotics and a viral vaccine, and an impaired-fidelity vaccine against SARS coronavirus.
You can find TWiV #207 at www.microbe.tv/twiv.