Initial observations about putative APOBEC3 deaminase editing driving short-term evolution of MPXV since 2017

[arambaut]

Initial observations about putative APOBEC3 deaminase editing driving short-term evolution of MPXV since 2017.

An updated version of this report is now available as a preprint at:
https://www.biorxiv.org/content/10.1101/2023.01.23.525187v1

This document is an initial report on the observation of an abundance of specific mutations in the 2022 MPXV outbreak and related virus genomes that can be ascribed to the action of APOBEC3 host enzymes. It should be considered work in progress and we plan to add additional analysis and interpretation. We also welcome discussion in the thread below. The analyses here are made possible by the groups and researchers who have shared MPXV genome sequence data (Table 1).

Áine O’Toole & Andrew Rambaut
Institute of Evolutionary Biology
University of Edinburgh
Edinburgh, UK

The first MPXV genome sequences from monkeypox cases in 2022 (Isidro et al. 2022; Selhorst et al. 2022) showed, phylogenetically, that these viruses had descended from a clade sampled in 2017-2019 from cases diagnosed in Singapore, Israel, Nigeria and the UK. Comparing 2022 genomes from Portugal, Belgium, USA, Australia, and Germany (see Table 1) with the closest earlier genomes (denoted UK_P2 and UK_P3), identified 47 shared single nucleotide differences (Figure 1).

The long term evolutionary rate of the related variola virus (VARV; the smallpox virus) has previously been estimated to be about 9x10^-6 (with 95% credible intervals of 7.8x10^-6 – 10.2x10^-6) substitutions per site per year (Firth et al. 2010) translating into about 1-2 nucleotide changes per year for a nearly 200,000 nucleotide genome. This makes 47 substitutions in the space of 3-4 years an unexpectedly large number. As MPXV is considered a zoonotic virus with limited human to human transmission, this long branch may be evidence of adaptation to humans allowing for the sustained transmission that is now observed.

However, 42 out of 47 of these nucleotide changes are of a particular type, a dinucleotide change from TC→TT or its reverse complement GA→AA. This specific mutation is characteristic of the action of the APOBEC3 family of deaminases. These act on single stranded DNA to deaminate cytosine to uracil causing a G→A mutation in the other strand when it is synthesised. Most human APOBEC3 molecules have a strong bias towards deaminating 5’TC dinucleotides , with the exception being APOBEC3G which prefers 5’CC dinucleotides (Yu et al. 2004).

Of the remaining 5 nucleotide changes between 2018 and 2022, 3 are GG→AG compatible with negative strand deamination and in the context preferred by APOBEC3G. A previous study showed that the related vaccinia virus was not significantly restricted by APOBEC3G (Kremer et al. 2006) and the low number of mutations at dimers preferred by this enzyme would seem to confirm this. The final 2 mutations are not the result of deamination of cytosine. Given the estimated rate of evolution for variola viruses between 2 and 5 substitutions would be within the expectation for 4 years of evolution. This could indicate that the 42 G→A and C→T changes were the result of bouts of deamination during virus replication due to host antiviral defences.

Genome sequencing of MPXV, including from archived samples and infections in non-human animals, has identified three distinct phylogenetic clades, although limited sampling means this may not be representative of the diversity present in the non-human animal reservoir host. The 2022 human outbreak is part of a clade that has previously been sampled in Nigeria or in cases with travel history in Nigeria with the oldest genome being from 1971 (accession number: KJ642617; (Nakazawa et al. 2015) with the remainder first sampled since 2017 (Mauldin et al. 2022). Examining the phylogenetic tree of these recent genomes also shows a predominance of the same GA→AA and TC→TT mutations suggesting that APOBEC3 deamination is the primary source of observed single nucleotide genetic variation in this group of MPXVs (Figure 1).

Figure 1 | A phylogenetic tree of MPXV genomes sampled from human infections from 2017-2022, with an outgroup sequence from an outbreak in Nigeria in 1971. SNPs along each branch are indicated with circles coloured by whether a mutation is putatively APOBEC edited TC→TT (blue) and GA→AA (orange), whether it is a non-target C→T or G→A mutation (teal), or other SNP type (grey).

This observation prompts a number of questions: Is this putative APOBEC3 editing occurring in a non-human animal reservoir host prior to emergence into humans in limited chains of human transmission? Or does this tree represent a multi-year history of sustained human transmission? Is the action of APOBEC3 acting as a driver of adaptation to humans as a host?

To address the first question we considered the wider context at which the putative deamination of cytosine was occurring (Figure 2) on the branch leading to the 2022 outbreak. Although there is some signal that T is not favoured immediately 3’ of the target cytosine, and G is not favoured to the 5’ of the target, there is not strong evidence of a wider preferential context. Studies have shown that the single APOBEC3 enzyme found in rodents exhibits a preference for a 5’TYC (Conticello et al. 2005), a context only seen for 46 of the 142 putative APOBEC3 substitutions across the entire tree.

Figure 2 | A) Observed heptamers that show a C→T or G→A mutated site in 44 of 47 mutations between the UK 2018 sequence and the CDC sequence of the current outbreak (accession numbers MT903345 and ON563414.2 respectively). B) Observed heptamers in 142 of 183 unambiguous mutations that show C→T or G→A mutated sites, normalised for strand across the entire tree excluding the mutations that occur on the 1971 branch. Heptamers associated with G→A mutations have been reverse-complemented to reflect deamination on the negative strand.

If APOBEC3 deamination is characteristic of replication in humans then we would expect to see very little evidence of it prior to the 2017 outbreak as this would primarily represent replication in the non-human reservoir. To examine this, we selected a further outgroup from Liberia, 1970 (accession number DQ011156.1; Likos et al. 2005) and identified 28 mutations that occur on the branch leading to the common ancestor of the 2017 MPXV genomes (Figure 3A). The mutations on this branch do not show such a strong signal, with only 10 of 28 SNPs matching the signature for APOBEC3 editing (Figure 3B). We therefore suggest that the pattern we see in these MPXV genomes since 2017 is indicative of replication in humans and the inheritance of the specific changes that occurred between 2017 and 2018 and then in the viruses from 2022 means that there has been sustained human to human transmission since at least 2017. The 10 mutations that do fit the APOBEC3 profile may represent an additional period of human to human transmission prior to the 2017 cases. Given that this is fewer APOBEC3 type mutations than seen in the branch between 2017 and 2018 (branch C in Figure 1), it is not likely that this represents a long period.

Figure 3 | SNPs that are unique to the branch leading to the common ancestor of the ingroup of the phylogeny in Figure 1 (2017 outbreak of MPXV) . A) Using an MPXV genome from Liberia, 1970 (accession number DQ011156; Likos et al. 2005) and the 1971 MPXV sequence (accession number KJ642617; Nakazawa et al. 2015) as outgroups, we identified mutations that occurred on the branch leading to the common ancestor of the 2017 MPXV genomes. These SNPs fit the mutational pattern described, with the two outgroup sequences sharing the same nucleotide sequence and the 2017 MPXV genomes sharing a distinct variant. B) Only 10 of the 28 mutations on this branch have the mutational signature of APOBEC editing.

Is APOBEC3 driving the fine-scaled evolution of MPXV in humans?

The normal action of APOBEC3 enzymes is anti-viral. Inducing C→T mutations at random locations across the genome is very likely to produce sufficient deleterious changes to inactivate the virus. Retroviruses seem to be a specific target of some APOBEC molecules (hA3G and hA3F) which can be packaged in the virion and then act as the reverse-transcriptase synthesises the first DNA strand from the RNA genome template (Harris et al. 2003; Zhang et al. 2003). The DNA copy is then integrated into the host genome replete with APOBEC induced mutations (G→A in the positive strand) meaning that all resulting progeny RNA genomes will have these mutations and will likely be lethally degraded. It is notable that HIV has a defence against APOBEC3 in a protein, vif, which degrades APOBEC3 and thus the effects are only seen in viruses where vif is defective resulting in ‘hypermutated’ viruses.

With double stranded DNA viruses APOBEC3 enzymes will act as the viral genome is being replicated and single strands are exposed. During repeated rounds of replication either strand can be deaminated leading to both C→T and G→A changes on the positive strand as seen here. Thus it is likely that the genomes that are extensively mutated by APOBEC3 will simply not be viable and will not be transmitted further. The extensive rounds of genome replication that MPXV undertakes in the cytoplasm may mean that most genomes are not affected by APOBEC3 action. Occasionally however a genome that is only modestally mutated by APOBEC3 may remain viable and be transmitted.

Considering all GA and TC dimer sites in the protein coding regions of the 2018 genome (‘MPXV-UK_P3’; accession number MT903345; Mauldin et al. 2022) – i.e., those that could be the target of APOBEC3 editing but had not been by that point – we looked at what the effect of a mutation at these sites would be (Figure 4). Of the 21,230 such dimers, 69.3% (14,707) would produce amino acid replacements, 24.7% (5253) would be synonymous, and 6.0% (1270) would induce stop codons. For the 2022 genomes, of the 40 mutations at these dimers that did occur, 60% (24) were amino acid replacements and 40% (16) were synonymous (a further 2 APOBEC3 mutations were in intergenic regions). The probability of getting 16 or fewer synonymous mutations under a binomial distribution with an expected rate of 0.247 is P=0.024. This supports the hypothesis that what we observe are the residual least harmful APOBEC3 mutations after natural selection has eliminated those with substantial fitness costs to the virus.

Figure 4 | A) Consequence of hypothetical APOBEC3 mutations at target dimer site (either the C in the TC target site or G in the GA target site) in currently unmutated sites in the 2018 MPXV genome (‘MPXV-UK_P3’; accession MT903345; Mauldin et al. 2022) and those observed APOBEC3 mutations on a representative sequence of the current outbreak (specifically, ‘MPXV_USA_2022_MA001’; accession ON563414; Gigante et al. 2022). These are categorised into synonymous (amino acid remaining unchanged), non-synonymous (altered amino acid) or nonsense (editing producing a stop codon).

Does the long branch represent adaptation to humans?

The ‘repertoire’ of mutations that APOBEC3 is able to provide as genetic variation on which natural selection can act is severely restricted. Only a limited number of dinucleotide contexts are present, and the amino acid changes that APOBEC3 editing can induce is also limited (Figure 5). Only 13 different amino acid replacements are possible and they are not reversible by the same mechanism. This means that the chance that a mutation that confers a benefit to the virus is amongst the ones available through APOBEC3 editing is relatively small.

Figure 5 | Amino acid mutations at AA and TT dimer sites in a reference MPXV genome (accession number JX878407) that would have occurred had unobserved APOBEC3 editing occurred to give rise to these. A) Barplot of amino acid changes categorised by Grantham Score (0-50 conservative, 51-100 moderately conservative, 101-150 moderately radical, >150 radical). B) Hypothetical amino acid changes had sites with AA and TT dimers had they been the result of APOBEC3 editing. Hypothetical mutations originating from stop codons excluded from analysis.

In most studies of APOBEC3 editing (and indeed, other host enzymes that induce mutations through deamination of nucleotides such as ADAR) the induced mutations are clustered and frequent, the presumption being that an APOBEC molecule when present repeatedly deaminates targets. For APOBEC3G in vif-defective HIV-1, it is estimated that only a few APOBEC molecules are packaged in the virion but induce large numbers of mutations (Armitage et al. 2008, 2012). Although there is some clustering of the observed mutations in the 2022 genomes they are generally seen across the entire genome (Figure 6). It is thus likely that this represents multiple episodes of APOBEC3 editing each of which had limited fitness consequences on the virus but conversely these mutations did not occur entirely independently. This non-independence may adversely affect the ability of molecular clock models to estimate time-calibrated phylogenetic trees. The non-reversibility of APOBEC3 induced mutations will also mean that certain phylogenetic substitution models will be more appropriate than others (i.e., non-reversible continuous time Markov chain models or Dollo model may be applicable).

Figure 6 | Distribution of mutational differences between UK_P3 and a representative genome from the 2022 outbreak.

A final consideration is that the constant pressure of APOBEC3 editing, despite MPXV being apparently largely robust to its antiviral effects in humans, may mean that moderately deleterious mutations may accumulate. This may result from genetic drift resulting from the diversity bottleneck at transmission. Given the directional nature of these mutations this may act as a ratchet, irreversibly accumulating fitness-reducing mutations with other mutational processes, occurring at a much lower rate, unable to overcome this. Indeed, additional APOBEC3 type mutations have been observed within 8 genomes sampled from the outbreak in Portugal (Isidro et al. 2022) including 2 that are shared by 2 genomes suggesting ongoing transmission of these.

The analysis presented here suggests further research including sequencing and analysis of within-host variation in APOBEC3 editing to investigate its potential for controlling or moderating MPXV human infections. Variation in APOBEC3 editing within a single individual (perhaps at samples from different lesions) might suggest that it is a significant burden on MPXV.

Table 1 | MPXV genome sequences used in this study.

Accession	Country	Year	Reference
KJ642617	Nigeria	1971	(Nakazawa et al. 2015)
DQ011156	Liberia	1970	(Likos et al. 2005)
MK783031	Nigeria	2017	(Yinka-Ogunleye et al. 2019)
MK783029	Nigeria	2017	(Yinka-Ogunleye et al. 2019)
MK783027	Nigeria	2017	(Yinka-Ogunleye et al. 2019)
MK783028	Nigeria	2017	(Yinka-Ogunleye et al. 2019)
MK783030	Nigeria	2017	(Yinka-Ogunleye et al. 2019)
MK783033	Nigeria	2017	(Yinka-Ogunleye et al. 2019)
MK783032	Nigeria	2017	(Yinka-Ogunleye et al. 2019)
MT903339	Nigeria	2017	(Mauldin et al. 2022)
MT903340	Nigeria	2017	(Mauldin et al. 2022)
MT903338	Nigeria	2017	(Mauldin et al. 2022)
MG693724	Nigeria	2017	(Faye et al. 2018)
MN648051	Israel	2018	(Cohen-Gihon et al. 2020)
MT903342	Singapore	2019	(Mauldin et al. 2022)
MT250197	Singapore	2019	(Yong et al. 2020)
MT903341	Nigeria	2018	(Mauldin et al. 2022)
MT903343	UK	2018	(Mauldin et al. 2022)
MT903344	UK	2018	(Mauldin et al. 2022)
MT903345	UK	2018	(Mauldin et al. 2022)
ON563414.2	USA	2022	(Gigante et al. 2022)
UZ-REGA-1	Belgium	2022	(Vanmechelen et al. 2022)
ON568298	Germany	2022	(Antwerpen et al. 2022)
PT0001 - PT0010	Portugal	2022	(Isidro et al. 2022)

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[m.koopmans]

really interesting, Aine, Andrew. However I do wonder about what this means. I know little about APOBEC editing, but understood there are examples of APOBEC editing in animals (fi mice, rats) for APOBEC1. Given that we do not really know the range of natural hosts of monkeypox, this may also be an option, I presume? There has been a general increase both in Lassa and monkeypox reports which have made me wonder whether there is a common ecological factor. Very speculative, could also all be explained by ascertainment bias, of course, but interested to hear your views

[m.koopmans]

to clarify, not referring to APOBEC1, but seeing for APOBEC1 similar mechanisms have been found in humans and rodents, assuming that could also be the case for APOBEC3.

[arambaut]

Hi Marion,
It is always possible that this is the action of a deaminase in another host species. But we are still seeing these TC->TT and GA->AA mutations occurring amongst the 2022 sequences so we can be sure it at least also happens in humans. It doesn’t seem to have been happening (at least to the same extent) in the deeper parts of the MPXV tree so is probably not going on in the original non-human host. So that leaves some jump to another host species prior to 2017 that has a similar APOBEC to humans, and then these cases are jumps from that host. Certainly not impossible but I think less parsimonious.

[gustavo_palacios]

@arambaut This is a very interesting hypothesis that would take a while to process and coalesce. However, I find there are a few things that we would need to explain before accepting as a signature of human-to-human transmission:

1. We know very little about the rodent reservoir genomic diversity and we do not know a lot either about the potential restrictions of MPXV regarding their host. The observation regarding the APOBEC different restriction in rodent vs primate is based on very little comparative genomics characterization. As for bats, their genomic diversity is immense. So, an alternative explanation would be that this strain would be a single spillover from a different rodent population that has driven that change. I have people in my lab looking into this, as we have been doing for bats before.

2. as I mentioned before (and you suggest), APOBEC3 signatures appear to be clustered and the deamination occurs indiscriminately in that area. With double-stranded DNA viruses, APOBEC3 enzymes will act as the viral genome is replicated and single strands are exposed. Thus, a good control would be to define areas of the pox genome that are likely to be “exposed” to APOBEC3 at the same time than mutated T-to-C/A-to-G sites (let’s say 500bp upstream and downstream) and find out the percentage of APOBEC target motifs that are “modified/unmodified” around.

3. APOBEC3 deaminase activity was not observed affecting Vaccinia (Vaccinia virus replication is not affected by APOBEC3 family members - PMC) but I think that the best control for your experiment would be to use smallpox datasets as they are clearly examples of human-to-human transmission.

4. I believe your analysis suffers also from an overestimation of the number of mutations as the 5 changes in the ITR (3 NS & 2 syn) will be increasing your confidence parameters, but are likely a biological or sequencing artifact.

My two cents.
Very interesting work!!! I think this is a very interesting observation that we need to keep exploring!

[arambaut]

Thanks Gus - yes all good points (although we do discuss some of them as caveats in the report). The point about the 5 linked mutations in the terminal repeats is important and in future we will consider them as non-independent. But we also warn about trying to use these mutations as any form of ‘molecular clock’.

With respect to clustering - I think we have to assume that the observed pattern is the result of multiple bouts of editing where APOBEC induced very few edits each time (thus not knocking out the virus). I believe the nature of the replication is that one strand is left progressively exposed as the polymerase works on the other strand before going round the hairpin and going back along. So that would mean there was a gradient of exposure time from one end to the other. But, I think this will be symmetrical so what we would end up with is the middle of the genome having the least exposure over all. That might accord with Figure 6, above, if you squint.

As we say in the piece, the Kremer paper only looks at APOBEC3G which we state is unlikely to be acting here because of its context preference for GG (or CC) which we don’t see here. It also doesn’t actually look for deamination editing - only growth restriction. It is also looking at vaccinia.

Finally - for other host species, as I say to Marion, above, it is entirely possible that there was another host switch (domesticated animal?) from which the human cases are coming which also has APOBEC3 with the same context. But we also see that exactly the same editing is still going on in the 2022 outbreak which we are pretty sure is human to human. So is another level of complexity to explain the pattern.

[maskin]

Only just beginning to take in this and all its possible implications but I had a comment about this statement: “For APOBEC3G in vif-defective HIV-1, it is estimated that only a few APOBEC molecules are packaged in the virion but induce large numbers of mutations (Armitage et al. 2008, 2012). Although there is some clustering of the observed mutations in the 2022 genomes they are generally seen across the entire genome (Figure 6 ). It is thus likely that this represents multiple episodes of APOBEC3 editing”. I’m not entirely clear about the line of argument here but I believe that APOBEC needs to be co-packaged in retrovirions because the cDNA synthesis occurs in the capsid after entry into the cytoplasm. In contrast, poxvirus dsDNA replication is understood to occur after the parental DNA is released from the core into the cytoplasm. Thus retroviruses would be modified by APOBECs from the ‘donor cell’, whereas poxviruses would be modified by APOBECs from the ‘recipient cell’. Whereas retrovirus modification will be restricted by the small numbers of APOBECs that are packaged, the poxvirus genome might be accessible to many more APOBECs from the new host’s cytoplasm. However that raises all sorts of questions: is the parental genome already partially sequestrated from the cytoplasm within the nascent virosome; are the relevant APOBECs induced by interferon (and is monkeypox virus able to control the human IFN response as well as it can probably control the rodent IFN response)?