Evidence linking APOBEC3B genesis and evolution of innate immune antagonism by gamma-herpesvirus ribonucleotide reductases
Viruses have evolved diverse mechanisms to antagonize host immunity such as direct inhibition and relocalization of cellular APOBEC3B (A3B) by the ribonucleotide reductase (RNR) of Epstein-Barr virus. Here, we investigate the mechanistic conservation and evolutionary origin of this innate immune counteraction strategy. First, we find that human gamma-herpesvirus RNRs engage A3B via largely distinct surfaces. Second, we show that RNR-mediated enzymatic inhibition and relocalization of A3B depend upon binding to different regions of the catalytic domain. Third, we show that the capability of viral RNRs to antagonize A3B is conserved among gamma-herpesviruses that infect humans and Old World monkeys that encode this enzyme but absent in homologous viruses that infect New World monkeys that naturally lack the A3B gene. Finally, we reconstruct the ancestral primate A3B protein and demonstrate that it is active and similarly engaged by the RNRs from viruses that infect humans and Old World monkeys but not by the RNRs from viruses that infect New World monkeys. These results combine to indicate that the birth of A3B at a critical branchpoint in primate evolution may have been a driving force in selecting for an ancestral gamma-herpesvirus with an expanded RNR functionality through counteraction of this antiviral enzyme.
This important work builds on the conceptual framework of host-pathogen interactions and co-evolution, adding new examples of co-divergence of primate herpesviruses with their respective host restriction factors. The authors convincingly outline the degree to which their initial findings (BORF2 and A3B interactions) are conserved across other herpesvirus RNRs, and place them in the context of the evolution of the A3 gene locus and expansion. This work will be of great interest to virologists. Especially those that work in the field of host pathogen evolution and the molecular arms race.https://doi.org/10.7554/eLife.83893.sa0
Cell-intrinsic innate immune proteins, or ‘restriction factors’, contribute to the first line of defenses against incoming viruses (reviewed by Duggal and Emerman, 2012). A prototypical example is the APOBEC3 (A3) family of single-stranded (ss)DNA cytosine deaminases, which provide broad and overlapping protection against viral infections (reviewed by Harris and Dudley, 2015; Simon et al., 2015). Classically, A3s exert antiviral activity by catalyzing cytosine deamination in exposed ssDNA replication intermediates, often resulting in lethal mutagenesis of viral genomes. Humans encode seven A3 enzymes (A3A-D, A3F-H), which have been implicated in the restriction of retroviruses (HIV and HTLV) (Sheehy et al., 2002; Harris et al., 2003; Wiegand et al., 2004; Sasada et al., 2005; Dang et al., 2006), human endogenous retroviruses (HERV) (Esnault et al., 2005; Lee et al., 2008), human hepadnaviruses (HBV) (Turelli et al., 2004; Suspène et al., 2005), human papillomaviruses (HPV) (Vartanian et al., 2008), polyomaviruses (BK) (Peretti et al., 2018), and most recently the gamma-herpesvirus Epstein-Barr virus (EBV; Cheng et al., 2019b). It is difficult to predict which subset of A3 enzymes has the potential to restrict a given virus, but differences in the subcellular localization properties of these enzymes provide useful insights into their antiviral targets. For instance, cytoplasmic A3s like A3G and A3F potently restrict HIV-1 by binding to viral genomic RNAs in the cytoplasm, packaging into budding virions at the cell membrane, and deaminating viral ssDNA intermediates during reverse transcription (Harris and Liddament, 2004; Mangeat et al., 2003; Navarro et al., 2005). It thus follows that A3B—the only constitutively nuclear human A3 enzyme—may pose a threat to the genomic integrity of viruses that undergo replication in the nucleus, such as EBV and related herpesviruses.
Viruses that are susceptible to restriction by cellular A3 enzymes have evolved equally potent counteraction mechanisms to ensure successful replication (reviewed by Harris and Dudley, 2015; Simon et al., 2015; Malim and Bieniasz, 2012). The most well-studied A3 antagonist is the lentiviral accessory protein Vif, which nucleates the formation of an E3 ubiquitin ligase complex to promote the selective degradation of up to five different cytoplasmic A3 enzymes: A3C, A3D, A3F, A3G, and A3H (reviewed by Harris and Dudley, 2015; Hu et al., 2021; Uriu et al., 2021a). Extensive mutagenesis analyses revealed that HIV-1 Vif evolved to use distinct binding surfaces in order to recognize different A3s, as underscored by separation-of-function mutants of Vif that selectively degrade only a subset of these enzymes (reviewed by Hu et al., 2021; Kitamura et al., 2011; Aydin et al., 2014). These studies have been essential to our understanding of how viruses adapt to overcome innate host defenses and, collectively, highlight the importance of elucidating structure-function relationships between proteins at the host-pathogen interface.
HIV-1 and related retroviruses are thought to be primary targets of A3 enzymes due to obligate ssDNA replication intermediates, abundant evidence for A3-catalyzed hypermutation of viral genomes, and a potent A3 counteraction mechanism. Reports of DNA viruses exhibiting signatures of A3-mediated mutagenesis suggest that these viruses may also be susceptible to restriction by A3 enzymes (Harris and Dudley, 2015; Shapiro et al., 2021), although the identification of viral antagonists has been challenging. We recently discovered that the large double-stranded (ds)DNA herpesvirus EBV uses a two-pronged approach to counteract the antiviral activity of A3B. The large subunit of the EBV ribonucleotide reductase (RNR), BORF2, directly inhibits A3B catalytic activity and relocalizes this enzyme from the nucleus to cytoplasmic aggregates—most likely to protect viral genomes from deamination during lytic replication (Cheng et al., 2019b). Consistent with this interpretation, BORF2-null EBV accumulates A3B signature C/G to T/A mutations upon lytic reactivation and exhibits significantly lower infectivity (Cheng et al., 2019b).
The deamination activity of all A3 family enzymes requires a zinc-coordinating motif, which forms the core of the catalytic site and is surrounded by three loop regions (L1, L3, and L7) that contribute to binding of ssDNA substrates (Shi et al., 2017; Kouno et al., 2017; Maiti et al., 2018). These loops vary in size and amino acid composition and account for differences in A3 catalytic rates and local target sequence preferences (reviewed by Silvas and Schiffer, 2019; Maiti et al., 2021). The neutralization of A3B by EBV BORF2 is the first example of direct inhibition of A3 ssDNA deaminase activity by another protein, and our recently published cryo-EM structure of EBV BORF2 in complex with the catalytic domain of A3B (A3Bctd) provides critical insights into the structural basis and molecular mechanism of this unanticipated host-pathogen interaction (Shaban et al., 2022). Specifically, our cryo-EM studies revealed that EBV BORF2 binds to the L1 and L7 regions of A3Bctd, resulting in a high-affinity interaction that effectively blocks the A3B active site from binding to ssDNA substrates and catalyzing deamination. Recently, we and others have shown that, in addition to EBV BORF2, the large RNR subunit from related human herpesviruses can also bind and relocalize A3B—as well as the highly similar A3A enzyme—suggesting that this A3 counteraction mechanism may be conserved evolutionarily (Stewart et al., 2019; Cheng et al., 2019a; Cheng et al., 2021).
Here, we investigate the molecular and functional conservation of A3 antagonism by the RNR subunits of different herpesviruses using structurally and evolutionarily informed approaches. We find that the RNR subunits of EBV and the related human gamma-herpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV) interact with A3B and A3A by binding to distinct loop regions near the catalytic site, which accounts for differences in binding selectivity and capacity to inhibit A3 enzymatic activity. We also uncover potential coevolutionary interactions between A3 enzymes and primate gamma-herpesviruses by discovering a link between the generation of the A3B gene in primates and the evolution of A3-neutralization functionalities in viral RNRs. Last, we reconstruct an ancestral primate A3B enzyme and show that it is bound, relocalized, and inhibited by the RNRs of gamma-herpesviruses that infect primate species that encode A3B but not by the RNRs from primate viruses that infect species that naturally lack this gene. Taken together, our data provide evidence of a conserved and likely ancient A3 antagonism mechanism in primate gamma-herpesviruses and offer insights into how genetic conflicts with host restriction factors may have affected—and likely continue to affect—the structure and function of viral proteins such as the herpesvirus RNR.
Structural differences and similarities between EBV and KSHV RNR large subunits
Our recent cryo-EM studies of EBV BORF2-A3B complexes revealed a large binding surface composed of multiple structural elements from each protein (Shaban et al., 2022). Specifically, loops 1 (L1) and 7 (L7) of the A3B catalytic domain (A3Bctd) are sequestered by multiple interactions with BORF2 including contacts with residues L133 and Y134 of a novel short helix insertion (SHI) and residues R484 and Y481 of an additional helical loop structure (HLS). Y134 additionally forms a network of stabilizing interactions with other BORF2 residues including Q476 and R484 (Shaban et al., 2022; depicted in Figure 1A). The BORF2 homolog from related gamma-herpesvirus KSHV (ORF61) has also been shown to bind A3B (Cheng et al., 2019b; Cheng et al., 2019a). However, an amino acid sequence alignment of EBV BORF2 and KSHV ORF61 indicates limited identity in the SHI and HLS regions (Figure 1B; complete sequence alignment in Figure 1—figure supplement 1A). We therefore used RoseTTAFold (Baek et al., 2021) to generate a structural model of KSHV ORF61 for three-dimensional comparisons (Figure 1C). Although sharing only ~42% primary amino acid identity, the predicted model of KSHV ORF61 overlays well with the cryo-EM structure of EBV BORF2 with root mean square deviation of 1.26 Å. Interestingly, in addition to similar HLS regions, EBV BORF2 and the predicted structure of KSHV ORF61 share a similar sized SHI region (depicted in red in Figure 1C), which is lacking in nonviral class Ia RNRs such as those from Escherichia coli and humans (Shaban et al., 2022).
To gain further insights into the potential role of KSHV ORF61 SHI and HLS regions in binding to A3B, we used in silico docking to investigate how ORF61 and A3Bctd might interact. RoseTTAFold models of KSHV ORF61 and A3Bctd were aligned to the cryo-EM BORF2-A3Bctd complex using PyMOL (DeLano, 2002) to create an initial docking pose followed by local protein-protein docking using RosettaDock 4.0 (Lyskov et al., 2013; Marze et al., 2018). Models were ranked by Critical Assessment of Predicted Interactions (CAPRI) score and the best structure was selected based on overall interface energy score (Materials and methods and Figure 1—figure supplement 1B). Several high-scoring models suggested that the ORF61 SHI region may interact with the loop 3 (L3) region of A3B, instead of L1 and L7 as for BORF2 (Figure 1D–E). Additionally, the predicted HLS region of KSHV ORF61 appears to be positioned further away from the RNR-A3Bctd binding interface and may not contribute to the interaction (Figure 1E). Taken together, these in silico predictions suggest that EBV BORF2 and KSHV ORF61 may differentially engage the catalytic domain of A3B by interacting with distinct loop regions. This possibility is tested in the following sections.
Involvement of the A3B and A3A loop 7 regions in binding to gamma-herpesvirus RNRs
Recent biochemical and structural studies demonstrated that direct contacts between EBV BORF2 and the L7 region of A3Bctd are essential for this host-pathogen interaction (Cheng et al., 2019b; Shaban et al., 2022). A3Bctd is closely related to A3A (>90% amino acid identity including identical L7 regions; Figure 2A), suggesting that a similar interaction with L7 could explain EBV BORF2 ability to engage A3B and A3A, but not other A3 family members. Recent work also revealed that KSHV ORF61 is similarly capable of binding and relocalizing A3B and A3A, but not other A3 enzymes (Cheng et al., 2019a).
To test whether binding to L7 is a conserved requirement for viral RNR-A3 interaction, we generated a series of chimeric A3 proteins with reciprocal L7 swaps and tested their ability to bind to EBV BORF2 and KSHV ORF61 in co-immunoprecipitation (co-IP) experiments (Figure 2B). 293T cells were transfected with FLAG-tagged viral RNRs and EGFP-tagged A3s, subjected to anti-FLAG affinity purification, and analyzed by immunoblotting. As expected from our prior studies, replacing L7 of A3Bctd or A3A with L7 of A3Gctd completely abolishes interaction with EBV BORF2 (Figure 2B, left panel, lanes 1–4) and, reciprocally, replacing L7 of A3Gctd with L7 of A3A/Bctd endows binding (Figure 2B, left panel, lanes 5–6). However, we were surprised to find that replacing L7 of A3Bctd or A3A with L7 of A3Gctd does not affect binding by KSHV ORF61 (Figure 2B, right panel, lanes 1–4). Accordingly, replacing L7 of A3Gctd with L7 of A3A/Bctd does not enable pull-down by KSHV ORF61 (Figure 2B, right panel, lanes 5–6).
An additional defining feature of the interaction between A3B/A and viral RNRs is relocalization from the nucleus to the cytoplasm (Cheng et al., 2019b; Stewart et al., 2019; Cheng et al., 2019a). To evaluate the subcellular localization phenotypes of the L7 chimeras described above, we performed quantitative fluorescence microscopy experiments using HeLa cells co-transfected with EGFP-tagged A3s and FLAG-tagged viral RNRs (Figure 2C, representative images). In agreement with our co-IP results, replacing L7 of A3Bctd or A3A with L7 of A3Gctd markedly decreases EBV BORF2-mediated relocalization, although low levels of A3Bctd L7G relocalization can still be detected in a subset of cells. Reciprocally, replacing L7 of A3Gctd with L7 of A3A/Bctd enables effective relocalization (Figure 2C, representative images; Figure 2D–E, quantification). In further agreement with our co-IP results, swapping L7 does not decrease KSHV ORF61-mediated relocalization of A3Bctd or A3A, nor does it increase A3Gctd relocalization (Figure 2C, representative images; Figure 2D–E, quantification). Collectively, our experimental data support the in silico protein docking predictions and indicate that, despite an overall high degree of predicted structural similarity, the RNR subunits from EBV and KSHV engage A3Bctd and A3A through at least partially distinct surfaces.
Role of A3B and A3A loop 3 in binding to gamma-herpesvirus RNRs
L3 is one of three loops that surround the A3 active site (Figure 2A). In our recent cryo-EM studies of the EBV BORF2-A3Bctd complex, we were unable to model A3Bctd L3 residues due to the flexibility of this region (Shaban et al., 2022; dashed region in Figure 3A). In silico protein-protein docking predictions in Figure 1D–E suggest that this loop may be important for KSHV ORF61 binding. Therefore, to examine whether L3 participates in viral RNR-A3 interactions, we performed co-IP experiments using a panel of chimeric A3 proteins with reciprocal L3 swaps (Figure 3B).
Once again, we observed markedly distinct phenotypes in co-IPs with EBV BORF2 and KSHV ORF61. For EBV BORF2, similar pull-down levels are observed for wild-type (WT) versus L3-swapped A3s (Figure 3B, left panel, lanes 1–4), whereas replacing L3 of A3Bctd or A3A with L3 of A3Gctd nearly abolishes the interaction with KSHV ORF61 (Figure 3B, right panel, lanes 1–4). Additionally, replacing L3 of A3Gctd with L3 of A3A/Bctd is sufficient to promote modest immunoprecipitation with KSHV ORF61 (Figure 3B, right panel, lanes 5–6). These results indicate that the L3 region is critical for KSHV ORF61 binding and likely to have little or no role in binding to EBV BORF2.
To evaluate the subcellular localization phenotypes of the L3 chimeras described above, we performed quantitative fluorescent microscopy experiments using HeLa cells (Figure 3C, representative images). Similar to our L7 results, microscopy observations are largely concordant with co-IP phenotypes. For EBV BORF2, L3 swaps have no substantial effect on A3 relocalization, whereas replacing L3 of A3Bctd or A3A with the corresponding region of A3Gctd causes a marked decrease in relocalization by KSHV ORF61 (Figure 3C, representative images; Figure 3D–E, quantification). Notably, KSHV ORF61 is able to relocalize the chimeric A3Gctd L3A/Bctd protein, albeit to considerably lesser extents compared to WT A3Bctd or A3A. These data provide further support for the in silico predictions above by demonstrating the importance of the L3 region for binding to KSHV ORF61 and promoting cytoplasmic aggregate formation in living cells.
Role of A3B and A3A loop 1 in binding to gamma-herpesvirus RNRs
An unanticipated discovery made possible by our recent EBV BORF2-A3Bctd cryo-EM structure is the existence of an extensive interaction between the short HLS region of BORF2 and L1 of A3Bctd (Shaban et al., 2022). This L1 interaction accounts for EBV BORF2 binding preferentially to A3B as opposed to A3A. We therefore compared the role of the L1 region of A3Bctd versus A3A in the interaction of these enzymes with EBV BORF2 and KSHV ORF61. First, we confirmed our recent report by showing that replacing L1 of A3A with L1 of A3Bctd leads to a marked increase in EBV BORF2 co-IP (Figure 4A, left panel, lanes 1–2). A strong increase in pull-down is also visible when L1 of A3A is replaced with L1 of A3Gctd, possibly due to the similarities between the L1 regions of A3Bctd and A3Gctd, where each enzyme has a 3-residue motif that is absent in A3A (PLV and PWV, respectively; Figure 2A). Reciprocally, replacing L1 of A3Bctd with L1 of A3A—but not with L1 of A3Gctd—dramatically diminishes the interaction with EBV BORF2 (Figure 4A, left panel, lanes 4–5).
In contrast, KSHV ORF61 does not appear to bind more strongly to A3Bctd compared to A3A (Figure 4A, right panel, compare lanes 1 and 4). Moreover, L1 swaps between A3Bctd and A3A have no effect on KSHV ORF61 co-IPs (Figure 4A, right panel, lanes 1, 2, 4, and 5). However, interestingly, replacing L1 of either A3Bctd or A3A with residues from A3Gctd decreases interaction with KSHV ORF61 (Figure 4A, right panel, lanes 3 and 6). This may be attributable to the presence of a unique bulky tryptophan residue in A3Gctd L1 potentially clashing with a nearby interacting surface (Figure 1 and Figure 2A).
We next assessed the subcellular localization phenotypes of these L1 chimeras using quantitative fluorescence microscopy (Figure 4B, representative images; Figure 4C–D, quantification). In contrast to the co-IP results above, swapping the L1 regions of A3Bctd and A3A does not overtly affect relocalization by EBV BORF2. This agrees with our published results (Shaban et al., 2022) and supports a model in which compensatory avidity interactions during cytoplasmic aggregate formation are likely to account for the similar relocalization phenotypes of A3Bctd and A3A despite BORF2 interacting more weakly with the latter protein. In comparison, for KSHV ORF61, swapping L1 of A3Bctd with L1 of A3Gctd slightly decreases relocalization, whereas the remaining L1 swaps have no significant effect (Figure 4B, representative images; Figure 4C–D, quantification).
EBV BORF2 interacts with A3Bctd loop 1 to inhibit DNA deaminase activity
In addition to causing protein relocalization, direct binding by EBV BORF2 potently inhibits A3B catalytic activity in vitro (Cheng et al., 2019b; Shaban et al., 2022). This supports a model in which EBV BORF2 simultaneously inhibits A3B activity and moves the complex away from viral replication centers to protect EBV genomes from deamination during lytic reactivation. Given results here showing that EBV BORF2 and KSHV ORF61 bind to both A3B and A3A, we next asked whether direct enzymatic inhibition is a conserved feature of this pathogen-host interaction.
HeLa T-REx cells stably expressing doxycycline-inducible A3B-EGFP or A3A-EGFP were transfected with an empty vector control or FLAG-tagged EBV BORF2 and KSHV ORF61. After 24 hr of doxycycline induction, whole-cell extracts were incubated with a fluorescently labeled ssDNA oligonucleotide substrate containing a single A3B/A deamination motif (5’-TC). A3B/A-mediated deamination of cytosine-to-uracil followed by uracil excision by UDG and abasic site cleavage by sodium hydroxide (NaOH) yields a shorter product that can be detected by SDS-PAGE and fluorescence scanning (see Materials and methods). As expected, EBV BORF2 strongly inhibits A3B deaminase activity in cell lysates as indicated by minimal product accumulation (Figure 5A, left panel, compare lanes 1 and 2). KSHV ORF61 also inhibits A3B-catalyzed deamination when compared to a no-RNR control, albeit to a considerably lesser extent compared to EBV BORF2 (Figure 5A, left panel, compare lanes 1 and 3).
Figure 5—source data 1
Figure 5—source data 2
Figure 5—source data 3
Surprisingly, neither EBV BORF2 nor KSHV ORF61 substantially inhibits the deaminase activity of A3A (Figure 5A, right panel). This stark difference is supported by our cryo-EM structure showing extensive contacts between BORF2 and the L1 and L7 regions of A3Bctd, which results in a high-affinity interaction that blocks the A3B active site from binding to ssDNA substrates and catalyzing deamination (Shaban et al., 2022). Specifically, A3B L1 residues 206-PLV-208 contribute to the formation of a hydrophobic pocket in which EBV BORF2 residue Y481 is inserted (depicted in Figure 5B). In contrast, this 3-residue motif is absent in A3A L1 (Figure 2A). We therefore hypothesized that inhibition of deaminase activity by EBV BORF2 requires not only binding to the catalytic domain through L7, but also a significant interaction with L1.
To further test this idea, we first generated mutants of EBV BORF2 Y481 and examined their ability to inhibit A3B-catalyzed deamination using the HeLa T-REx A3B-EGFP system described above. Mutating EBV BORF2 Y481 to an alanine or a proline effectively prevents inhibition of A3B-catalyzed deamination, likely by weakening the interaction with the L1 region (Figure 5C). Next, we compared the ability of EBV BORF2 to inhibit the deaminase activity of WT versus L1 swapped A3B and A3A in HeLa cells transfected with FLAG-tagged BORF2 and EGFP-tagged A3s. In agreement with results from Figure 5A, WT A3B deaminase activity is potently inhibited by EBV BORF2 (Figure 5D, left, lanes 1 and 2). In contrast, when L1 of A3B is replaced with L1 of A3A, EBV BORF2 completely loses its capacity to inhibit ssDNA deamination (Figure 5D, left, lanes 3 and 4). In further agreement with data in Figure 5A, WT A3A deaminase activity is not inhibited by EBV BORF2 (Figure 5D, right, lanes 1 and 2). Unfortunately, the chimeric A3A L1B construct exhibits very low levels of ssDNA deaminase activity, and it is therefore not possible to determine the susceptibility to inhibition by EBV BORF2 (Figure 5D, right, lanes 3 and 4). Nevertheless, these results combine to indicate that the EBV BORF2 interaction with A3B L1 is a requirement for inhibiting deamination and also help explain why A3A activity is not inhibited similarly.
A structural overlay of the cryo-EM structure of the EBV BORF2-A3B complex and the RoseTTAFold-predicted model of KSHV ORF61 indicates that a residue analogous to the interlocking Y481 of BORF2 is lacking in ORF61 (Figure 5E). Instead, KSHV ORF61 has a proline (P488) immediately downstream of shared proline and threonine residues. This difference suggests that L1 may be allowed to remain flexible and engage ssDNA substrates when A3B is bound to KSHV ORF61. Alternatively—and perhaps more likely—a lack of interaction with L1 may result in RNR-A3B complexes with lower binding affinity (i.e., lower association rate and/or higher dissociation rate), thereby rendering KSHV ORF61 unable to inhibit A3B-catalyzed deamination to the same extent as EBV BORF2.
The genesis of A3B may have shaped gamma-herpesvirus RNR evolution in primates
A3 genes are unique to placental mammals and the evolutionary history of the A3 locus is marked by frequent expansions and contractions resulting from independent events of gene duplication, loss, and fusion (Münk et al., 2012; LaRue et al., 2008; Ito et al., 2020; Conticello, 2008). For instance, in the primate lineage leading to humans the A3 locus evolved from an ancestral 3 deaminase domain cluster to the present-day 11 domain (7 gene) cluster. Interestingly, phylogenetic and comparative genomics studies indicate that A3B may be the youngest A3 family member in primates (LaRue et al., 2008; Uriu et al., 2021b). Specifically, A3B was likely generated by the duplication of ancestral deaminase domains in the common ancestor of Catarrhini primates (i.e., hominoids and Old World monkeys) and is consequently absent in Platyrrhini primates (i.e., New World monkeys) (depicted in Figure 6A). We therefore hypothesized that the function of an ancestral gamma-herpesvirus RNR as an innate immune antagonist may have been selected by the birth of A3B during this critical period of primate evolution (i.e., 43–29 million years ago, after the split of Catarrhini and Platyrrhini primates).
To test this idea, we cloned the RNR large subunit genes from representative gamma-herpesviruses that infect primates with A3B (EBV and KSHV, which infect humans, and McHV-4 and McHV-5, which infect rhesus macaques) and related viruses that infect primates without A3B (CalHV-3 and SaHV-2, which infect New World marmosets and squirrel monkeys, respectively) (Figure 6B). Of note, these RNRs represent gamma-herpesviruses from two evolutionarily diverged genera—Lymphocryptovirus and Rhadinovirus (Figure 6B). We then examined the capacity of these viral RNRs to bind to human A3Bctd and A3A in co-IP experiments (Figure 6C). In support of our hypothesis, only the RNRs from viruses that infect A3B-encoding primates (humans and rhesus macaques) pull-down human A3Bctd and A3A (Figure 6C, lanes 1–4, left and right panels). In contrast, the RNRs from viruses that infect primates without A3B (marmosets and squirrel monkeys) are unable to pull down either enzyme (Figure 6C, lanes 5–6, left and right panels).
We next asked if these viral RNRs can relocalize human A3B and A3A. HeLa T-REx cells were transfected with FLAG-tagged viral RNRs and incubated with doxycycline to induce A3B-EGFP or A3A-EGFP expression (Figure 6D). In agreement with our co-IP results, only the RNRs from viruses that infect A3B-encoding primates are able to relocalize human A3B and A3A from the nucleus to the cytoplasmic compartment. Together, these results suggest that the ability to bind and relocalize human A3 enzymes is conserved among RNRs from gamma-herpesviruses that infect Catarrhini primates.
We additionally used HeLa T-Rex cells stably expressing doxycycline-inducible human A3B-EGFP or human A3A-EGFP to ask whether these viral RNRs are capable of inhibiting the ssDNA deaminase activity of human A3B and A3A (Figure 6E). Consistent with our previous results, EBV BORF2 potently inhibits A3B (Figure 6E, top panel, lane 2). A similarly strong inhibition is seen with the RNR of rhesus macaque Lymphocryptovirus McHV-4 (Figure 6E, top panel, lane 3). The RNRs from KSHV and rhesus macaque Rhadinovirus McHV-5 did not overtly affect the formation of a product band but appeared to slow down A3B-catalyzed deamination, as suggested by a stronger substrate band compared to the no-RNR control (Figure 6E, top panel, lanes 5 and 6). Most importantly, the RNRs from marmoset Lymphocryptovirus CalHV-3 and squirrel monkey Rhadinovirus SaHV-2 are both defective in inhibiting A3B activity (Figure 6E, top panel, lanes 4 and 7). Additionally, none of the six different viral RNRs inhibits the activity of human A3A (Figure 6E, bottom panel). Collectively, our results suggest a possible link between the birth of A3B in the common ancestor of Catarrhini primates and the evolution of A3B-counteraction functionalities in an ancestral gamma-herpesvirus RNR large subunit (i.e., direct binding, subcellular relocalization, and enzymatic inhibition).
A short HLS from EBV BORF2 enables the marmoset CalHV-3 RNR to bind to human A3B and A3A
A3 enzymes are composed of single or double zinc-coordinating (Z) domains that can be grouped into three classes (Z1, Z2, and Z3) based on sequence similarity (LaRue et al., 2009). In humans, A3A, A3C, and A3H are single Z domain proteins (Z1, Z2, and Z3, respectively), whereas A3B, A3G, A3D, and A3F harbor two Z domains (Z2-Z1 for A3B and A3G, Z2-Z2 for A3D and A3F). Although New World monkeys lack A3B, the genomes of these species—as well as all primates—encode a single-domain Z1 enzyme homologous to human A3A. Therefore, it is possible that the RNRs of gamma-herpesviruses that infect New World monkeys may have evolved to antagonize the A3A enzymes of their natural host species.
To investigate this possibility, we performed co-IP experiments using FLAG-tagged viral RNRs and host-matched EGFP-tagged A3As (Figure 7A). In line with our results above with human A3Bctd and A3A (Figure 6), all four RNRs from viruses that infect Catarrhini primates bind to the A3A enzymes of their respective host species (Figure 7A, lanes 1, 2, 4, and 5). In contrast, the RNRs from viruses infecting New World monkeys are unable to bind to their hosts’ A3As (Figure 7A, lanes 3 and 6). We next asked if these RNRs can relocalize the A3A enzymes encoded by their respective host species (Figure 7B). In agreement with the co-IP results, the RNRs from EBV/KSHV and McHV-4/McHV-5 relocalize human and rhesus A3A, respectively (Figure 7B, left and center). In contrast, the RNRs from CalHV-3 and SaHV-2 do not alter the localization of marmoset and squirrel monkey A3A, respectively (Figure 7B, right). These results further support the idea that A3 antagonism by gamma-herpesvirus RNRs is likely to be limited to viruses that infect primates with A3B and, additionally, suggest that binding to A3A may be a consequence of similarity to A3Bctd (identical loop 3 and loop 7 regions and >90% overall identity).
Our recent cryo-EM structure of the EBV BORF2-A3Bctd complex revealed that loops 1 and 7 of A3B are sequestered by multiple interactions with BORF2 including contacts with a short HLS involving the critical BORF2 residues Y481 and R484 (Shaban et al., 2022; region depicted in red in Figure 7C and alternative poses in Figure 1 and Figure 5). Amino acid sequence alignments of the RNRs from human EBV, rhesus macaque McHV-4, and marmoset CalHV-3, which are all members of the Lymphocryptovirus genus, show that the former two RNRs share identical amino acid sequences in the HLS region, whereas the latter protein has multiple differences (HLS region alignment in Figure 7C). Given the overall similarity between the three-dimensional architecture of EBV BORF2 and the predicted structure of CalHV-3 RNR (Figure 7—figure supplement 1), we next asked whether replacing the HLS region of the CalHV-3 RNR with the corresponding motif from EBV BORF2 may be sufficient to enable binding to A3B/A enzymes in co-IP experiments. Interestingly, although the chimeric CalHV-3 RNR construct is still unable to bind marmoset A3A (Figure 7D, lane 2), this protein is now able to efficiently bind to both human A3Bctd and A3A (Figure 7D, lanes 3 and 4). These results indicate that the HLS region of EBV BORF2 is sufficient to endow a normally non-binding Lymphocryptovirus RNR with a capacity to engage both human A3Bctd and A3A, likely by enabling a direct physical interaction with the identical loop 7 region of these enzymes (L7 region alignment in Figure 7E).
Ancestral A3B is active and antagonized by present-day RNRs from gamma-herpesviruses that infect A3B-encoding primates
To gain deeper insights into the interaction between A3B and gamma-herpesvirus RNRs, we generated a phylogenetic tree of all publicly available primate A3B sequences and used the GRASP-suite (Foley et al., 2022) to reconstruct the amino acid sequence of ancestral A3B (Figure 8A and Figure 8—figure supplement 1). We then examined an expanded panel of primate gamma-herpesvirus RNRs (i.e., 11 different viral proteins) for a capacity to bind human and ancestral A3Bctd in co-IP experiments (Figure 8B). In agreement with results above, only the RNRs from gamma-herpesviruses that infect primates with A3B are capable of binding to human A3Bctd (Figure 8B, left). Similarly, nearly all RNRs from viruses that infect primates with A3B are able to bind the ancestral protein and, importantly, all viruses that infect primates which naturally lack the A3B gene are unable to bind the ancestral enzyme (Figure 8B, right).
To further explore the properties of the ancestral A3B protein, we performed a comprehensive series of subcellular localization experiments and ssDNA deaminase activity assays using this expanded panel of primate gamma-herpesvirus RNRs (Figure 8C–D and Figure 8—figure supplement 2A). As above, human A3B is completely relocalized from the nucleus to the cytoplasmic compartment by the RNRs of viruses that infect humans and other primates with an A3B gene (Figure 8C, top; Figure 8—figure supplement 2A). Similar results were obtained with ancestral A3B, although only partial relocalization is observed with the KSHV ORF61 (Figure 8C, bottom; Figure 8—figure supplement 2A). KSHV ORF61 also exhibit a decreased ability to inhibit the deaminase activity of ancestral A3B compared to human A3B (Figure 8D, compare lane 5 from the top and bottom panels). These relocalization and ssDNA deaminase activity results correlate with the co-IP observations in Figure 8B, where the RNRs from the two human gamma-herpesviruses showed decreased binding to ancestral A3Bctd compared to the human enzyme. Such differences in binding to human versus ancestral A3B may be an indication of adaptive changes in present-day viral RNRs driven by host-pathogen conflicts.
A phylogenetic tree based on the amino acid sequences of available gamma-herpesvirus RNRs indicates that the sequences from human, rhesus macaque, and marmoset Lymphocryptoviruses cluster together and the sequences from the different Rhadinoviruses cluster according to viral lineage (Figure 8E). Interestingly, despite its similarity to KSHV ORF61, the RNR from ColHV-1—a novel KSHV-related virus identified in African Colobinae monkeys (Dhingra et al., 2019)—differs from the RNRs of all other viruses that infect Catarrhini primates tested here in that it shows no ability to bind, relocalize, or inhibit present-day human or ancestral A3B (Figure 8B, Figure 8—figure supplement 2A, and Figure 8D) or interact with its host’s A3A (Figure 8—figure supplement 3A). This apparent incongruity may be explained by the fact that African Colobinae monkeys appear to have lost the A3B gene following the Colobinae subfamily split into the African and Asian tribes 10–14 million years ago (Figure 8—figure supplement 3B–C), which presumably relieved the pressure for ColHV-1 to maintain an A3B-neutralization activity.
Differential binding of viral RNRs to present-day and ancestral A3B is likely a reflection of past—and possibly still ongoing—genetic conflicts. To further explore this idea, we analyzed a dataset of primate A3B DNA sequences for evidence of codon sites under diversifying/positive selection using the mixed effects model of evolution algorithm (MEME) (Murrell et al., 2012). We detected evidence for diversifying selection at multiple sites, including clusters of positively selected residues in loops 1 and 3 (Figure 8F, top; Figure 8—figure supplement 4A–B). This suggests that the amino acid composition of these regions may have been influenced by recurrent diversifying selection, possibly as a mean to escape binding by gamma-herpesvirus RNRs, which in turn would select for virus adaptation to restore binding (depicted in Figure 8F, bottom). In line with this interpretation and together with results in Figure 3 and Figure 4, it is possible that differences in positively selected residues in loops 1 and 3 may respectively account for the apparent increased ability of EBV BORF2 and KSHV ORF61 to antagonize present-day human A3B compared to ancestral A3B.
Viruses use a plethora of mechanisms to avoid and antagonize host immune responses. We previously reported an unexpected role for herpesvirus RNRs in counteracting innate immunity by revealing that EBV repurposes its RNR subunit BORF2 to neutralize the antiviral activity of cellular A3B (Cheng et al., 2019b). The importance of this A3B counteraction mechanism is evidenced by BORF2-null EBV exhibiting lower infectivity and an accumulation of A3B signature C/G-to-T/A mutations (Cheng et al., 2019b). Subsequent studies revealed that the RNR subunits from related human herpesviruses also bind and relocalize A3B, as well as the highly similar A3A enzyme (Stewart et al., 2019; Cheng et al., 2019a). Here, we leverage knowledge gained from our recent cryo-EM structure of EBV BORF2 in complex with the catalytic domain of A3B (Shaban et al., 2022) to investigate the mechanistic conservation of A3 antagonism by herpesviruses. We find that EBV BORF2 and KSHV ORF61 bind to cellular A3 enzymes via distinct surfaces and show that direct inhibition of ssDNA deaminase activity by EBV BORF2 requires specific interactions with the L1 of A3B in addition to the L7 region. We further find that across different primate gamma-herpesvirus lineages, the existence of an RNR-mediated mechanism to counteract cellular A3 enzymes appears to be confined to viral species that infect A3B-expressing primates. Together, these results suggest that the genesis of A3B by duplication of ancestral Z2 and Z1 deaminase domains in the common ancestor of present-day hominoids and Old World monkeys may have selected for an ancestral gamma-herpesviruses with a capacity to antagonize this new restriction factor through its large RNR subunit. To our knowledge, this is the first study to link the birth of an antiviral host factor and the evolution of an equally potent viral escape mechanism.
Our data here not only highlight the overall conservation of A3B antagonism by gamma-herpesvirus large RNR subunits but also reveal new and unexpected molecular differences. In agreement with our recent cryo-EM studies (Shaban et al., 2022), we show that A3B L7 is a critical determinant of EBV BORF2 binding, whereas A3B L1 serves to help strengthen this interaction and confer selective binding over A3A (Figure 2 and Figure 4). Surprisingly, these same rules do not apply to KSHV ORF61, which appears to mainly rely on L3 to bind and relocalize cellular A3B/A (Figure 3). Together, our results suggest a model where interactions with L7 and/or L3—which are identical in A3B and A3A, but not in any other members of the A3 family—enable gamma-herpesvirus RNRs to bind and relocalize both enzymes, whereas additional interacting regions (i.e., EBV BORF2 HLS and A3B L1) may help confer selectivity and additional neutralization activity (e.g., potent inhibition of A3B ssDNA deaminase activity). It is additionally interesting that this may be a new manifestation of a pathogen-host wobble mechanism (Richards et al., 2015) in which ongoing small changes over evolutionary time in the viral RNRs can manifest molecularly in the present-day target protein as distinct binding surfaces (e.g., EBV BORF2 requires A3Bctd L7 and KSHV ORF61 requires L3).
Using a panel of 11 primate gamma-herpesvirus RNRs, we show that, in addition to binding and relocalization, all RNRs derived from viruses that infect hosts that encode A3B show some capacity to inhibit the ssDNA deamination activity of human A3B, but none of these viral proteins could inhibit the deaminase activity of human A3A (Figure 8 and Figure 8—figure supplement 2B). This is consistent with a model in which the antiviral activity of A3B may have forced these viruses to evolve and maintain a potent counteraction mechanism, whereas interaction with A3A may be a consequence of sequence homology to A3B and less likely a true physiologic function. This interpretation is further supported by our observation that the RNRs from viruses that infect A3B-null primates are incapable of interacting with the A3A enzymes of their natural host species (Figure 7).
The A3B-selective nature of this host-pathogen interaction is orthogonally supported by previous binding studies showing that the EBV BORF2-A3Bctd interaction has a biolayer interferometry dissociation constant of ~1 nM, whereas the interaction with the >90% identical A3A enzyme is over 10-fold weaker (Shaban et al., 2022). Our results here reveal that, in addition to providing selective binding to A3B, EBV BORF2 interaction with the L1 region of A3B is required for potently inhibiting DNA deaminase activity (Figure 5). It is thus possible that the direct binding of EBV BORF2 to two loop regions near the A3B catalytic site—L1 and L7—helps stabilize the BORF2-A3B complex, resulting in a high affinity interaction and consequently blocking the A3B active site from accessing ssDNA substrates and catalyzing deamination. Importantly, the potential adaptability of this A3B counteraction mechanism in Lymphocryptoviruses is highlighted by results showing that grafting the HLS region from EBV BORF2—which contains residues that directly contact A3B L1 and L7—into the RNR from marmoset CalHV-3 enables binding to human A3B (Figure 7). In comparison, KSHV ORF61 binding to A3B largely through L3 may result in a lower affinity interaction that more weakly inhibits ssDNA deamination. These inferences support a model in which binding to the A3 active site by viral RNRs is responsible for both protein relocalization and enzymatic inhibition, yet substantial deaminase activity inhibition is only observed above a certain affinity threshold. We therefore postulate that preferential binding to A3B might have been an evolutionary adaptation resulting from selective pressures disproportionally affecting gamma-herpesviruses within the Lymphocryptovirus genus, such as EBV and McHV-4. For instance, EBV latency is limited to B lymphocytes (Young and Rickinson, 2004), which can express high levels of endogenous A3B (Koning et al., 2009; Refsland et al., 2010), strongly implying that the neutralization of this enzyme is required to preserve viral genome integrity upon lytic reactivation. On the other hand, the broader tropism of KSHV suggests that lytic replication might not always occur in the presence of A3B (Blasig et al., 1997; Caselli et al., 2005; Wu et al., 2006; Kim et al., 2003; Chakraborty et al., 2012), which may result in lower exposure to A3B-mediated deamination.
Evolutionary analyses of host-pathogen antagonistic relationships are powerful tools for understanding protein functions. The link between A3B generation by duplication of ancestral Z2 and Z1 domains and the evolution of A3-neutralization functionalities in herpesvirus RNRs described here suggests that the double domain architecture of A3B may be a requirement for herpesvirus restriction. Notably, the non-catalytic Z2 domain of A3B has been shown to account for this enzyme’s distinct nuclear localization (Salamango et al., 2018) and association with multiple RNA binding proteins, in addition to allosterically regulating the catalytic activity of the Z1 domain (Xiao et al., 2017). It is therefore plausible that cooperation between Z2 and Z1 domains accounts for A3B antiviral function, possibly by facilitating access to herpesvirus transcription complexes inside the nucleus (e.g., ssDNA exposed in viral R-loop structures; McCann et al., 2021). Future studies dissecting the molecular mechanisms that allow A3B to recognize and target foreign nucleic acids such as herpesvirus genomes might reveal insights into the regulation of ssDNA deaminase activity and how the loss of such regulatory mechanisms can at times result in cellular chromosomal DNA mutations and cancer development (reviewed by Venkatesan et al., 2018; Olson et al., 2018). Future work will likely benefit from interrogating the RNRs and A3 enzymes from a larger and more diverse panel of virus and host species, which is currently prevented by the relatively small number of herpesvirus complete genomes deposited in public databases. Bat species may be particularly interesting given they encode the largest known A3 repertoire in mammals (Hayward et al., 2018; Jebb et al., 2020) and are also known to host a variety of different herpesviruses (Wibbelt et al., 2007; Watanabe et al., 2010; Wu et al., 2012; Anthony et al., 2013; Sasaki et al., 2014; Sano et al., 2015; Zheng et al., 2016; Shabman et al., 2016; Escalera-Zamudio et al., 2016; James et al., 2020; Letko et al., 2020).
Materials and methods
The large RNR subunit from EBV (HHV-4), McHV-4, CalHV-3, KSHV (HHV-8), McHV-5, McHV-11, McHV-12, AtHV-3, SaHV-2, ColHV-1, and SgHV-1 were obtained as gBlocks purchased from Integrated DNA Technologies and cloned into pcDNA4/TO (Invitrogen #V102020) with a C-terminal 3×FLAG tag (DYKDDDDK) by restriction digestion and ligation. EBV BORF2Y481A, EBV BORF2Y481P, CalHV-3 RNRWEARDV490YDSRDM constructs were generated by site-directed mutagenesis using Q5 High-Fidelity DNA Polymerase (NEB #M0491). WT constructs match the following GenBank accessions: EBV BORF2 (V01555.2), McHV-4 BORF2 (AY037858.1), CalHV-3 ORF55 (AF319782.2), KSHV ORF61 (U75698.1), McHV-5 ORF61 (AF083501.3), McHV-11 ORF61 (AY528864.1), McHV-12 ORF61 (KP265674.2), AtHV-3 ORF61 (AF083424.1), SaHV-2 ORF61 (X64346.1), ColHV-1 ORF61 (MH932584.1), and SgHV-1 ORF26 (OK337614.1).
APOBEC3 enzymes were obtained as gBlocks purchased from Integrated DNA Technologies and cloned into pcDNA5/TO (Invitrogen #V103320) with a C-terminal EGFP-tag by restriction digestion and ligation. The loop swap constructs A3B_L1A3A25-30, A3Bctd193-382_L1A3A25-30, A3Bctd193-382_L1A3G209-217, A3Bctd193-382_L3A3G247-254, A3Bctd193-382_L7A3G317-322, A3A_L1A3B205-213, A3A_L1A3G209-217, A3A_ L3A3G247-254, A3A_L7A3G317-322, A3Gctd197-384_L3A3A60-67, and A3Gctd197-384_L7A3A132-137 were generated by site-directed mutagenesis using Q5 High-Fidelity DNA Polymerase (NEB #M0491). WT constructs match the following GenBank accessions: Homo sapiens A3B (NM_004900), Homo sapiens A3A (NM_145699), Homo sapiens A3G (NM_021822), Macaca mulatta A3A (NM_001246231.1), Macaca mulatta A3B (NM_001246230.2), Callithrix jacchus A3A (NM_001301845.1), Saimiri boliviensis A3A (XM_039462978.1), Colobus angolensis palliatus A3A (XM_011964212.1).
Human cell culture
Unless indicated, cell lines were derived from established laboratory collections. All cell cultures were supplemented with 10% fetal bovine serum (Gibco #26140-079), 1× penicillin-streptomycin (Gibco #15140-122), and periodically tested for mycoplasma contamination using MycoAlert PLUS Mycoplasma Detection Kit (Lonza #LT07-710). 293T cells were cultured in high glucose DMEM (HyClone #SH30022.01) and HeLa cells were cultured in RPMI 1640 (Gibco #11875-093).
To generate HeLa T-REx A3A-EGFP and A3B-EGFP cell lines, HeLa cells were transduced with pLENTI6/TR (Invitrogen #V480-20), selected with 5 μg/ml blasticidin (GoldBio #B-800-25), and subcloned by serial dilution to obtain single cell clones. Clonal populations were stably transfected with pcDNA5/TO-A3A-EGFP or pcDNA5/TO-A3B-EGFP, selected with 200 μg/ml hygromycin (GoldBio #H-270-1), and subcloned by serial dilution. Representative clones were used for DNA deaminase activity assays (Figure 5A, Figure 5C, Figure 6E) and IF microscopy experiments (Figure 6D).
Approximately 250,000 293T cells were seeded in six-well plates and transfected the next day with 100 ng pcDNA5/TO-A3-EGFP and 200 ng pcDNA4/TO-RNR-3×FLAG using TransIT-LT1 (Mirus #2304). Cells were harvested ~24 hr after transfection and resuspended in 500 µl of lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10% glycerol, 0.5% IGEPAL, 100 μg/ml RNaseA, 1 tablet of Sigma-Aldrich cOmplete Protease Inhibitor Cocktail). Resuspensions were vortexed, sonicated on ice-bath for 10 s at the lowest setting using a Branson sonifier, and centrifuged at 15,000 × g at 4°C for 15 min. A 50 µl aliquot was removed from each sample for input detection and the remaining clarified whole-cell lysate was incubated with 20 μl packed gel volume of anti-FLAG M2 magnetic beads (Sigma-Aldrich #M8823) at 4°C overnight with gentle rotation. Samples were washed three times with 500 µl of lysis buffer and bound protein was eluted with 50 µl of elution buffer (100 µg/ml FLAG peptide [Sigma-Aldrich #F3290], 50 mM Tris-HCl pH 7.4, 150 mM NaCl). Input and elution samples were analyzed by immunoblot using mouse anti-GFP (1:5000, Clontech #632381) to detect A3-EGFP, rabbit anti-FLAG (1:5,000, Sigma-Aldrich #F7425) to detect RNR-3×FLAG, and rabbit anti-GAPDH (1:5000, Proteintech #60004) to detect GAPDH (loading control). Secondary antibodies used were IRDye 800CW anti-rabbit (LI-COR #926-32211) and HRP-linked anti-mouse (Cell Signaling #7076), both at 1:10,000 dilution.
Immunofluorescence microscopy experiments
In Figure 6D, approximately 5000 engineered HeLa T-REx cells (described above) were seeded into 96-well imaging plates (Corning #3904) and transfected the next day with 30 ng pcDNA4/TO-RNR-3×FLAG. A3B-EGFP or A3A-EGFP expression was induced 6 hr after transfection with 50 ng/ml doxycycline. Approximately 24 hr after induction, cells were fixed in 4% formaldehyde (Thermo Fisher #28906), permeabilized in 0.2% Triton X-100 (Sigma-Aldrich #T8787) for 10 min and incubated in blocking buffer (2.8 mM KH2PO4, 7.2 mM K2HPO4, 5% goat serum (Gibco #16210064), 5% glycerol, 1% cold water fish gelatin (Sigma-Aldrich #G7041), 0.04% sodium azide, pH 7.2) for 1 hr. Cells were then incubated with primary antibody mouse anti-FLAG (1:1000, Sigma #F1804) overnight at 4°C with gentle rocking to detect FLAG-tagged viral RNRs. The next day, cells were incubated with secondary antibody anti-mouse Alexa Fluor 594 (1:1000, Invitrogen #A-11005) for 2 hr at room temperature protected from light and nuclei were counterstained with Hoechst 33342 (Thermo Fisher #62249) for 10 min. Representative images were acquired using a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek) at ×20 magnification.
In Figures 2—4, approximately 5000 HeLa cells were seeded into 96-well imaging plates (Corning #3904) and transfected the next day with 15 ng pcDNA5/TO-A3-EGFP and 30 ng pcDNA4/TO-RNR-3×FLAG. Cells were fixed approximately 24 hr after transfection. Permeabilization, antibody staining, and nuclei counterstaining with Hoechst 33342 were performed as described above. Plates were imaged using a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek) at ×20 magnification using the automated imaging function to capture 50 non-overlapping images per well. Background subtraction was performed using Fiji (Schindelin et al., 2012) and images were analyzed using CellProfiler v4.2 (Stirling et al., 2021b). A pipeline was generated to identify individual nuclei, trace cell boundaries, segment the cytoplasmic/nuclear compartments, and take measurements of intensity, texture, and correlation for the Hoechst (nuclear stain), RNR, and A3 channels within each subcellular compartment. At least 100 cells were measured for each well. All measurements were exported to an SQLite database and CellProfiler Analyst v3.0 (Stirling et al., 2021a) was used for scoring phenotypes by machine learning in order to quantify the number of cells showing relocalized versus non-relocalized A3 within the population of RNR-expressing cells in each well. The percentages of cells with relocalized A3 in Figure 2D, Figure 3D, and Figure 4C were calculated for n=3 independent experimental replicates (i.e., data generated from the analysis of cells seeded, transfected, and imaged on three different days). The ridgeline plots in Figure 2E, Figure 3E, and Figure 4D were generated in RStudio with the ggplot2 package (Wickham, 2016) using the Pearson correlation coefficient measurements for the A3 and Hoechst channels within each segment cell nucleus.
In Figure 7B and Figure 8C, approximately 5000 HeLa cells were seeded into 96-well imaging plates (Ibidi #89626) and transfected the next day with 15 ng pcDNA5/TO-A3-EGFP and 30 ng pcDNA4/TO-RNR-3×FLAG. Cells were fixed approximately 24 hr after transfection. Permeabilization, antibody staining, and nuclei counterstaining with Hoechst 33342 were performed as described above. Plates were imaged using an Eclipse Ti2-E inverted microscope (Nikon) at ×20 magnification.
ssDNA deaminase activity assays
In Figure 5A, Figure 5C, and Figure 6E, approximately 250,000 HeLa T-REx cells stably expressing doxycycline-inducible A3B-EGFP or A3A-EGFP (described above) were seeded into six-well plates and transfected the next day with 200 ng pcDNA4/TO-RNR-3×FLAG using TransIT-LT1 (Mirus #2304). A3B-EGFP or A3A-EGFP expression was induced 6 hr after transfection with 50 ng/ml doxycycline. Approximately 24 hr after induction, cells were harvested, resuspended in 100 µl of reaction buffer (25 mM HEPES, 15 mM EDTA, 10% glycerol, 1 tablet of Sigma-Aldrich cOmplete Protease Inhibitor Cocktail), and subjected to freeze-thaw. Whole-cell lysates were then centrifuged at 15,000 × g for 15 min and the clarified supernatant was transferred to a new tube. Soluble lysates were incubated with 100 µg/ml RNAse A at room temperature for 1 hr. A 5 µl aliquot was removed from each sample for immunoblots using mouse anti-GFP (1:5000, Clontech #632381) to detect A3-EGFP, rabbit anti-FLAG (1:5000, Sigma-Aldrich #F7425) to detect RNR-3×FLAG, and rabbit anti-GAPDH (1:5000, Proteintech #60004) to detect GAPDH (loading control). Secondary antibodies used were IRDye 800CW anti-rabbit (LI-COR #926-32211) and HRP-linked anti-mouse (Cell Signaling #7076), both at 1:10,000 dilution. Five µl of soluble lysate were incubated at 37°C for 1 hr with 0.7 µM of a fluorescent ssDNA substrate (5′-ATT ATT ATT ATT CAA ATG GAT TTA TTT ATT TAT TTA TTT ATT T-fluorescein-3′) and 2.5 U of UDG (NEB #M0280) in a total reaction volume of 10 µl (diluted in reaction buffer). NaOH was then added to the reaction mix to a final concentration of 100 mM and samples were incubated at 98°C for 10 min. The reaction was then mixed with 11 µl 2× formamide buffer (80% formamide, 1×TBE, bromophenol blue, and xylene cyanol) and reaction products were separated on a 15% TBE-urea PAGE gel. Separated DNA fragments were visualized on a Typhoon FLA 7000 scanner (GE Healthcare) on fluorescence mode.
In Figure 5D and Figure 8D, approximately 250,000 293T cells were seeded in six-well plates and transfected the next day with 100 ng pcDNA5/TO-A3-EGFP and 200 ng pcDNA4/TO-EBV-BORF2-3×FLAG. Cells were harvested approximately 24 hr after transfection and the DNA deaminase activity assay proceeded as described above.
Protein structure modeling
Protein structure models were generated with RoseTTAFold (Baek et al., 2021) using the ROBETTA server followed by simple all-atom refinement of the top-ranked models using the Relax application in the ROSIE 2 server (Lyskov et al., 2013). To model the structure of the ORF61-A3Bctd complex (Figure 1), RoseTTAFold-predicted models of KSHV ORF61 and A3Bctd were aligned to the cryo-EM BORF2-A3Bctd complex (pdb: 7RW6) using PyMOL (DeLano, 2002) to arrange the starting docking pose and local protein-protein docking was performed using the RosettaDock 4.0 server (Lyskov et al., 2013; Marze et al., 2018). The 10,000 generated structures were sorted based on CAPRI score (Lensink et al., 2020) and subsequently ranked according to their interface energy score (Figure 1—figure supplement 1B). All protein structure figures were made using PyMOL (DeLano, 2002).
Herpesvirus RNR phylogenetic trees generation
Amino acid sequences of the large RNR subunits from the following herpesviruses were obtained from the NCBI Protein RefSeq database: HSV-1 (YP_009137114.1), HSV-2 (YP_009137191.1), VZV (NP_040142.1), EBV (YP_401655.1), HCMV (YP_081503.1), HHV-6A (NP_042921.1), HHV-6B (NP_050209.1), HHV-7 (YP_073768.1), KSHV (YP_001129418.1), McHV-4 (YP_067953.1), CalHV-3 (NP_733909.1), McHV-5 (NP_570809.1), McHV-11 (AAT00096.1), McHV-12 (AJE29717.1), AtHV-3 (AAC95585.1), SaHV-2 (NP_040263.1), ColHV-1 (QDQ69272.1), and SgHV-1 (UNP64460.1). To generate the phylogenetic trees in Figure 6B and Figure 8E, protein sequences were aligned using MUSCLE (Edgar, 2004) and maximum likelihood phylogenies were inferred using PhyML 3.0 (Guindon et al., 2010) using the Smart Model Selection (SMS) tool (Lefort et al., 2017) and 100 bootstraps.
Ancestral APOBEC3B sequence reconstruction
To generate the phylogenetic tree in Figure 8A, primate A3B amino acid sequences were aligned using MUSCLE (Edgar, 2004; Figure 8—figure supplement 1) and a maximum likelihood phylogeny was inferred using PhyML 3.0 (Guindon et al., 2010) using the SMS tool (Lefort et al., 2017) and 100 bootstraps. Reconstruction of the ancestral A3B protein sequence was performed with GRASP (Foley et al., 2022) using the sequence alignment and phylogenetic tree above.
Positive selection analysis
Primate A3B DNA sequences were aligned using ClustalOmega (Sievers et al., 2011) and a maximum likelihood phylogeny was inferred using PhyML 3.0 (Guindon et al., 2010) using the SMS tool (Lefort et al., 2017) and 100 bootstraps. Codon sites under episodic diversifying selection were identified with the HyPhy 2.5 software package (Pond and Muse, 2005) using the MEME (Murrell et al., 2012) with 100 bootstraps and p-value threshold of 0.1 (Figure 8—figure supplement 4A–B).
All data generated and analyzed during this study are included in the manuscript and supporting files. Numerical data for the bar plots and ridgeline plots in Figure 2, Figure 3, and Figure 4 are provided in Source data 1. Source Data Files containing uncropped gel images and raw files for immunoblots and deaminase activity assays are provided for Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 8-figure supplement 2, and Figure 8-figure supplement 3. GenBank codes for the sequences used in this study are listed in the Materials and Methods section.
Kaposi’s sarcoma associated herpesvirus entry into target cellsFrontiers in Microbiology 3:6.https://doi.org/10.3389/fmicb.2012.00006
Ensembl 2022Nucleic Acids Research 50:D988–D995.https://doi.org/10.1093/nar/gkab1049
Identification of APOBEC3DE as another antiretroviral factor from the human APOBEC familyJournal of Virology 80:10522–10533.https://doi.org/10.1128/JVI.01123-06
SoftwareThe pymol molecular graphics systemPymol.
Novel virus related to Kaposi’s sarcoma-associated herpesvirus from colobus monkeyEmerging Infectious Diseases 25:1548–1551.https://doi.org/10.3201/eid2508.181802
Evolutionary conflicts between viruses and restriction factors shape immunityNature Reviews. Immunology 12:687–695.https://doi.org/10.1038/nri3295
Muscle: multiple sequence alignment with high accuracy and high throughputNucleic Acids Research 32:1792–1797.https://doi.org/10.1093/nar/gkh340
Retroviral restriction by APOBEC proteinsNature Reviews. Immunology 4:868–877.https://doi.org/10.1038/nri1489
APOBECs and virus restrictionVirology 479–480:131–145.https://doi.org/10.1016/j.virol.2015.03.012
Differential evolution of antiretroviral restriction factors in pteropid bats as revealed by APOBEC3 gene complexityMolecular Biology and Evolution 35:1626–1637.https://doi.org/10.1093/molbev/msy048
Multifaceted HIV-1 Vif interactions with human E3 ubiquitin ligase and apobec3sThe FEBS Journal 288:3407–3417.https://doi.org/10.1111/febs.15550
Novel herpesviruses in neotropical bats and their relationship with other members of the herpesviridae familyInfection, Genetics and Evolution 84:104367.https://doi.org/10.1016/j.meegid.2020.104367
Defining APOBEC3 expression patterns in human tissues and hematopoietic cell subsetsJournal of Virology 83:9474–9485.https://doi.org/10.1128/JVI.01089-09
TimeTree: a resource for timelines, timetrees, and divergence timesMolecular Biology and Evolution 34:1812–1819.https://doi.org/10.1093/molbev/msx116
A novel gamma 2-herpesvirus of the rhadinovirus 2 lineage in chimpanzeesGenome Research 11:1511–1519.https://doi.org/10.1101/gr.158601
Guidelines for naming nonprimate APOBEC3 genes and proteinsJournal of Virology 83:494–497.https://doi.org/10.1128/JVI.01976-08
Hypermutation of an ancient human retrovirus by APOBEC3GJournal of Virology 82:8762–8770.https://doi.org/10.1128/JVI.00751-08
Virus taxonomy: the database of the International Committee on taxonomy of viruses (ICTV)Nucleic Acids Research 46:D708–D717.https://doi.org/10.1093/nar/gkx932
Sms: smart model selection in phymlMolecular Biology and Evolution 34:2422–2424.https://doi.org/10.1093/molbev/msx149
Modeling protein‐protein, protein‐peptide, and protein‐oligosaccharide complexes: CAPRIProteins: Structure, Function, and Bioinformatics 88:916–938.https://doi.org/10.1002/prot.25870
Bat-borne virus diversity, spillover and emergenceNature Reviews. Microbiology 18:461–471.https://doi.org/10.1038/s41579-020-0394-z
Interactions of apobec3s with DNA and RNACurrent Opinion in Structural Biology 67:195–204.https://doi.org/10.1016/j.sbi.2020.12.004
Hiv restriction factors and mechanisms of evasionCold Spring Harbor Perspectives in Medicine 2:a006940.https://doi.org/10.1101/cshperspect.a006940
Apobec enzymes as targets for virus and cancer therapyCell Chemical Biology 25:36–49.https://doi.org/10.1016/j.chembiol.2017.10.007
BookHyPhy: hypothesis testing using phylogeniesIn: Pond S, editors. Statistical Methods in Molecular Evolution. Springer. pp. 125–181.https://doi.org/10.1093/bioinformatics/bti079
APOBEC3B nuclear localization requires two distinct n-terminal domain surfacesJournal of Molecular Biology 430:2695–2708.https://doi.org/10.1016/j.jmb.2018.04.044
Isolation and characterization of a novel alphaherpesvirus in fruit batsJournal of Virology 88:9819–9829.https://doi.org/10.1128/JVI.01277-14
Fiji: an open-source platform for biological-image analysisNature Methods 9:676–682.https://doi.org/10.1038/nmeth.2019
Structural basis for targeted DNA cytosine deamination and mutagenesis by APOBEC3A and APOBEC3BNature Structural & Molecular Biology 24:131–139.https://doi.org/10.1038/nsmb.3344
APOBEC3s: DNA-editing human cytidine deaminasesProtein Science 28:1552–1566.https://doi.org/10.1002/pro.3670
Intrinsic host restrictions to HIV-1 and mechanisms of viral escapeNature Immunology 16:546–553.https://doi.org/10.1038/ni.3156
Two distinct lineages of macaque gamma herpesviruses related to the Kaposi’s sarcoma associated herpesvirusJournal of Clinical Virology 16:253–269.https://doi.org/10.1016/s1386-6532(99)00080-3
Elucidation of the complicated scenario of primate APOBEC3 gene evolutionJournal of Virology 95:e00144-21.https://doi.org/10.1128/JVI.00144-21
Novel betaherpesvirus in batsEmerging Infectious Diseases 16:986–988.https://doi.org/10.3201/eid1606.091567
Novel Kaposi’s sarcoma-associated herpesvirus homolog in baboonsJournal of Virology 77:8159–8165.https://doi.org/10.1128/jvi.77.14.8159-8165.2003
Discovery of herpesviruses in batsThe Journal of General Virology 88:2651–2655.https://doi.org/10.1099/vir.0.83045-0
BookGgplot2: Data AnalysisSpringer.
Virome analysis for identification of novel mammalian viruses in bat species from Chinese provincesJournal of Virology 86:10999–11012.https://doi.org/10.1128/JVI.01394-12
Structural determinants of APOBEC3B non-catalytic domain for molecular assembly and catalytic regulationNucleic Acids Research 45:7494–7506.https://doi.org/10.1093/nar/gkx362
Epstein-Barr virus: 40 years onNature Reviews. Cancer 4:757–768.https://doi.org/10.1038/nrc1452
Jan E CaretteReviewing Editor; Stanford University School of Medicine, United States
Sara L SawyerSenior Editor; University of Colorado Boulder, United States
In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.
[Editors' note: this paper was reviewed by Review Commons.]https://doi.org/10.7554/eLife.83893.sa1
We would like to thank all three reviewers for their time and thorough assessment of our manuscript. We appreciate their constructive feedback and believe our work has been considerably strengthened by addressing the comments, suggestions, and concerns raised during peer review. In the following responses, we address the reviewer’s critiques point-by-point.
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
Moraes et al. build upon their recent studies of APOBEC3 antagonism by EBV BORF2 by showing that additional RNR subunits encoded by other herpesviruses share this activity, suggesting that the host-virus arms races involving APOBEC3 proteins is more widespread than previously thought. Furthermore, the authors show that herpesviruses infecting primates that lack A3B (New World Monkeys) do not apparently exhibit a capacity to antagonize human A3B, suggesting that this function was not required during the evolution of those viruses (while it seemingly was important for viruses infecting hosts that encode A3B). Overall, this is a technically sound submission that combines confocal immunofluorescence, co-immunoprecipitation, and enzymatic assays to comprehensively test the sensitivity of different A3s to counteraction by viral RNRs. The enzymatic (deamination) assays performed prove to be the most insightful, since co-IP and colocalization microscopy was not entirely sufficient to reveal which domains of A3 are important for targeting by RNRs. It is well-written, well-organized, and well-referenced, and will be of interest to readers who study APOBEC3s, herpesviruses, and host-virus arms races more generally.
Thank you for appreciating the technical aspects and broader impact of our studies.
1. Figure 4B: the fluorescence microscopy data does not match well with the Co-IP (in Figure 4A). For example, L1B in A3A enhances the A3A-BORF2 co-IP but no clear differences are observed in colocalization. Or are the authors claiming the presence of L1B results in greater colocalization between A3A and BORF2, because there is slightly less diffuse BORF2 in the cytoplasm under these conditions? If that is the case, then quantitative colocalization analysis will need to be performed. In general, virtually none of the colocalization analysis in Figure 4B matches well with the co-IP results of Figure 4A. The authors take this to suggest that L7, and not L1, is most important determinant for BORF2 binding to A3s, but in that case, then the colocalization data is disconnected functionally from co-IP results. This is not necessarily a large problem, since the authors ultimately test the enzymatic activity of A3s in the presence of different RNRs. These latter functional experiments more objectively define what regions of A3s are important for antagonism by RNRs.
Thank you for giving us an opportunity to clarify these important points. We understand how the fluorescence microscopy data in Figure 4B may at first appear to disagree with the co-IP results in Figure 4A. However, we would like to point out that WT A3A—which has a shorter L1 region and binds less strongly to BORF2 compared to A3B—is nevertheless efficiently relocalized by BORF2 (PMID 35476445, 31534038, 31493648). We believe that this observation can be explained by compensatory avidity interactions during cytoplasmic aggregate formation in living cells, a process mediated by the formation of a non-canonical BORF2-BORF2 dimer as detailed in our recent cryo-EM studies (PMID 35476445). These avidity interactions explain how a weaker interaction (as indicated by weaker co-IP levels) can still result in the formation of large cytoplasmic aggregates. We have therefore revised our text to explain this apparent incongruity (page 11, lines 10-13).
2. Can the authors discuss/cite more about the actual subcellular compartments that the A3s are being relocated towards by the RNPs? In general, the authors' comments are limited to whether the A3 is predominantly in the nucleus, or not.
Previous imaging studies with markers for cytoplasmic organelles by our lab suggested that BORF2-A3B aggregates accumulate within the endoplasmic reticulum (ER) (PMID 30420783). However, in our recent cryo-EM studies of the BORF2-A3B complex (PMID 35476445), we discovered that disrupting BORF2-BORF2 dimerization prevents aggregate formation but does not affect EBV BORF2’s ability to bind to A3B and relocalize the complex to the cytoplasmic compartment. In other words, dimerization-deficient mutants of BORF2 clearly cause A3B-BORF2 heterodimers to appear diffusely cytoplasmic. Therefore, we no longer have a reason to implicate the ER and we aim to clarify this in future studies aiming to define the full molecular composition of the large cytoplasmic aggregates.
3. Since the authors draw a connection between the absence of A3B in New World Monkeys and the fact that New World Monkey-specific viruses don't seem to counteract A3s, can the authors discuss what could be learned by studying human individuals who lack A3B and the evolution of herpesviruses in those individuals?
This is a very interesting point, but we would prefer not to speculate on this in our manuscript. Although there is indeed an A3B deletion allele in the human population (predominantly southeast Asia), its worldwide allele frequency is quite low and most people still have 1 or 2 copies of this antiviral gene. Thus, the deletion allele frequency is not high enough to remove the selective pressure on the virus to maintain A3B counteraction activity through its RNR.
However, we did discover one Old World monkey species that completely lacks A3B (Colobus angolensis). We showed that the RNR from the γ-herpesvirus that infect these monkeys (ColHV-1) lacks the ability to antagonize human A3B, ancestral A3B, human A3A, or the endogenous A3A of its natural host (Figure 8 and Figure 8—figure supplement 3). Thus, as you predicted, relieving the selective pressure within a species over an evolutionary period of time likely resulted in loss of A3B-antagonism activity by the viral RNR (page 19, lines 8-16).
1. I'm not sure it makes sense to call out Figures 1A-D in the Introduction section, rather than the Results section.
We have changed the Introduction and removed the original Figure 1 completely. We have however added a new Figure 1, which provides a structural rationale for our overall experimental approach.
Reviewer #1 (Significance (Required)):
This work represents a step-wise advance from the authors' previous work on herpesvirus RNPs and counteraction of host APOBEC3s. I study host-virus molecular arms race on evolutionary scales and this article is of interest and significance to me, and I assume to others in the field as well. The findings found within the submission are interesting but not necessarily informative about human health and disease. However, the subsequent work that this manuscript inspires is likely to tell us more about herpesvirus evolution in human patients and the mechanisms by which APOBEC3s promote cancer.
We thank you again for appreciating the broader significance of our work and how the results present here may inspire important future studies.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Summary: Building off the groups prior work on A3B and EBV BORF2 interactions, here they have expanded their studies to examine additional herpesvirus RNRs, demonstrating which features are conserved. Using a combination of IP experiments and IF, they have included KSHV ORF61 and HSV-1 ICP6 RNRs, and demonstrated that the A3 loop structures, L1, L3, and L7 from A3A, A3B, and A3G play varying roles in determining the ability to interact with the different RNRs. They then go on to demonstrate that the ability of BORF2 to block the deaminase activity of A3B is dependent on the tyrosine at position 481. Lastly, and most interestingly, they show that RNRs from Old World monkeys, but not New World monkeys, can bind to A3A and A3B, lead to their re-localization, and block deaminase activity.
We thank you for appreciating the molecular details and broader impact of our studies. Please note that we have revised the paper to focus on γ-herpesviruses by removing the less informative results with HSV-1 and adding new studies on Old/New World viral RNRs including comparisons with ancestral A3B.
Major comments: The vast majority of this work is very convincing. The authors claims are clearly reflected in the data presented for the most part. However, the work done with HSV-1 ICP6 co-IP is not very convincing. The authors claim that L7 and L3 swaps from A3Bctd to A3Gctd decreases pulldown (lines 5-12, p.7; lines 18-21, p.8; line 17, p.16). The figures (2A, 3A, 4A) however show only A3A being pulled down with ICP6. The re-localization data however does seem more consistent with the above claims. The authors note this in line 9, p.8. However, they come to a different conclusion in line 2, p.8, regarding the discrepancy between IP and IF data.
As mentioned above, we have removed the less convincing results with HSV-1 ICP6. We believe uncovering the mechanistic details of HSV-1 ICP6 interaction with A3B will require significant additional work and, therefore, would prefer to address this question in future studies.
The data and methods are clearly presented, with the exception of the supplemental figures, where it is unclear how the predicted modeling was conducted.
We apologize for the brief description in our earlier submission. We have revised our Methods section and included a more detailed description regarding the generation of protein structural models (page 29, lines 20-23; page 30, lines 1-6).
Experiments all seem to be sufficiently replicated.
The references to prior studies seem comprehensive. Text and figures were all very clear. Introducing the supplemental figure 1 earlier, may provide clarity to the argument about degree of relatedness (line 2, p.7).
We agree with this suggestion and have made changes to introduce the structural model of KSHV ORF61 in our new Figure 1.
The suggestion of ORF61 interaction with L3 as an anchor region (line 10-12, p.9) was not very clear/could benefit from a bit more elaboration.
We agree with this comment and have placed the predicted structural model of KSHV ORF61 bound to A3Bctd in our new Figure 1 and we have changed the text to clarify the role of A3B L3 in binding to KSHV ORF61 (page 10, lines 4-8).
Reviewer #2 (Significance (Required)):This work builds on the conceptual framework of host-pathogen interactions and co-evolution, adding new examples of co-divergence of primate herpesviruses with their respective host restriction factors. Following up on past findings (Cheng et al., 2019; Shaban et al., 2021), and reports from others (Stewart et al., 2019), they outline the degree to which their initial findings (BORF2 and A3B interactions) are conserved across other herpesvirus RNRs, and place them in the context of the evolution of the A3 gene locus and expansion.
This work will be of great interest to virologists. Especially those that work in the field of host pathogen evolution and the molecular arms race.
My background is in host-pathogen interactions and herpesvirus evolution. I lack the sufficient expertise to evaluate the predicted modeling.
We thank you again for appreciating the novelty and significance of our work. We are also hopeful that it will be of great interest to virologists.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
In this manuscript, Moraes and colleagues build upon previous publications from this group to (1) characterize the variation in the ability of orthologs of BORF2 from six different herpesviruses to bind and/or relocalize and/or inhibit the deaminase activity of A3A and A3B; (2) use swaps and other mutagenesis to measure whether various regions and amino acids in A3A, A3B, and A3G contribute to the observed variation in the ability of RNR subunits from different viruses to bind these A3s.
The data convincingly show that different regions of different A3s contribute differently to binding of RNRs from different viruses. These same regions also have variable effects on RNR-mediated relocalization and inhibition of the deamination activity of A3A, A3B, and A3G. In the last set of experiments presented in the manuscript, the authors show that the RNR from the four viruses isolated from humans and rhesus macaques are able to bind human A3B, while the RNRs from two New World monkey viruses are unable to bind human A3B. Finally, the authors suggest a correlation between the timing of the birth of A3B in the branch leading to the last common ancestor of hominoids/Old World monkeys and the gain of A3 binding/antagonism by herpesvirus RNRs. However, these evolutionary implications are not convincingly supported by the current datasets and would require a significant burden of initial experiments to test.
We thank the reviewer for the nice summary of our work and for appreciating the loop swaps experiments showing differential RNR binding to APOBEC3s. In our original submission, we compared the RNRs of 4 viruses infecting Catarrhini primates and 2 viruses infecting New World primate species. We found that only the RNRs from viruses that infect Catarrhini primates bind, relocalize, and inhibit human A3B. We have now performed additional experiments to further investigate this remarkable association (Figures 7, 8, and associated supplementary material) which are detailed in our revised manuscript and summarized below:
First, we have expanded the scope of our experiments to include all publicly available RNR sequences from primate γ-herpesviruses (i.e., 11 RNRs in contrast to our initial 6 RNRs). Second, we tested this whole panel against human A3B and found that only the RNRs from viruses that infect Old World primates that encode A3B are able to bind, relocalize, and inhibit human A3B (Figures 6 and 8). In comparison, binding to human A3A in co-IP experiments is invariably weaker and/or not detectable, relocalization phenotypes are less pronounced, and DNA deaminase activity is not inhibited (Figure 6 and Figure 8—figure supplement 2B). Third, a subset of this RNR panel was tested against the A3A enzymes of their natural host species (Figure 7) and, again, only the RNRs form viruses that infect Old World primates bind and relocalize the A3A enzymes tested. Fourth, as an addition test of this idea, we grafted a short helical loop structure (HLS) from EBV BORF2 into the marmoset CalHV-3 RNR and showed that this small change enabled the chimeric protein to bind to both human A3B and A3A (likely through L7), though not to the natural marmoset A3A protein. Fifth, we used all available present-day primate A3B sequences to reconstruct the most likely ancestral A3B sequence and showed that this enzyme is nuclear and as active (if not more active) than human A3B (Figure 8). This ancestral A3B protein is also bound, relocalized, and inhibited by most present day RNRs from γ-herpesviruses that infect species with A3B, but not by the RNRs of any of the NWM-infecting viruses tested. The only exception to this association between A3B and Catarrhini-infecting γ-herpesviruses is the RNR of the African Colobus virus ColHV-1, which we found can likely be explained by the loss of A3B in its host species due to a deletion that occurred approximately 10-14 mya after the split of the Colobinae subfamily into African and Asian tribes, which further supports the idea that A3-antagonism by γ-herpesvirus RNRs is maintained by the selective pressure imposed by the antiviral activity of A3B.
1) The use of only the human orthologs of A3A and A3B limit the inferences that can be made regarding the ability of RNRs from various viruses to bind the A3s from the host species of that virus. For example, human A3A (and other hominoid A3As) have a rather distinct Loop 1 sequence, where that same loop in rhesus A3A is a much more similar to A3B. It follows that the RNRs from rhesus-tropic viruses could very well bind and inhibit A3A from rhesus. Likewise, the A3B-RNR interactions within and between species could differ markedly. Indeed, we know that the loops of A3s are some of the most rapidly evolving regions of these genes.
We agree fully with these points and have addressed them through several new experiments. We have now tested the RNRs from rhesus macaque and NWM γ-herpesviruses against the A3A enzymes of their natural host species (Figure 7) and found that only RNRs form viruses that infect Old World primates bind and relocalize the A3A enzymes tested. In addition, we used all available present-day primate A3B sequences to reconstruct the most likely ancestral sequence and showed that this ancestral A3B enzyme is antagonized exclusively by the RNRs of present-day γ-herpesviruses that infect A3B-encoding primates.
2) If RNR's ability to bind A3s correlated or was driven by the birth of A3B in catarrhine primates, the evolution of the binding/antagonism trait would be highly unparsimonious. The most parsimonious scenario would be emergence of A3 antagonism in the LCA of α and gammaherpesviruses (since the authors show A3 binding in HSV-1 and several gammaherpesviruses) with a loss of the trait in NWM-infecting viruses; alternatively, the trait could have been horizontally transferred gained 3 independent times, but this is certainly unlikely and not supported by any data. However, it is also possible that the RNRs from NWM infecting viruses do, in fact, bind/antagonize the A3 orthologs from NWMs. This needs to be tested before addressing the complexities of the birth of the antagonism trait.
Please see our responses above. All of our results support a model in which the birth of A3B in an ancestral primate selected for a γ-herpesvirus with A3B binding and neutralization activity and that this activity has been maintained through evolution and still manifests today in all of the tested present-day RNRs of γ-herpesviruses that infect species with A3B.
1) The authors should state more discreetly what is new to this paper and what was shown in previous paper and in some cases repeated here. For example, figure 1 is all repeated experiments from previous papers which is unusual for a manuscript.
This is a fair point and we have removed the original Figure 1 and replaced it with a structural model that provides a strong rationale for the rest of our studies. For the sake of clarity, we have also revised our text and made the necessary changes to ensure a clear distinction between new and repeated results.
2) The authors conclude that RNRs bind to A3s via partially distinct surfaces, but they don't actually test binding. Swaps and mutations do not show that the site of mutation is a site of interaction. but they do test the requirement of these AAs or regions for binding. Formally, these mutations could be exerting an allosteric effect on the binding interface of RNR and A3. In combination with the CryoEM data, these new data do support the model that these are different surfaces of interaction, but the wording should be more precise to present this.
In our revised manuscript we use a combination of in silico protein structure prediction and docking to model the binding interface between human A3Bctd and KSHV ORF61 (new Figure 1). This approach predicts an interaction with the L3 region of A3B, which we validate through co-IP and co-localization experiments (Figure 3). In contrast, EBV BORF2 requires the L7 region to bind to A3Bctd and this interaction is additionally strengthened by L1 residues (Figures 2 and 4). Taken together with our prior cryo-EM data, these results point to a model in which EBV BORF2 and KSHV ORF61 bind to different surfaces of A3B (albeit near the active site and likely due to evolutionary “wobbling”). We therefore believe it to be unlikely that this mechanism is allosteric given our prior structural studies and the likely common evolutionary origin of this A3B antagonism mechanism.
3) Similar to point 1, the authors repeatedly discuss the "most critical determinant of EBV BORF2 binding" and other "most critical" interactions. This is not supported by the data and should be changed to something along the lines of 'the site of largest effect among the sites we analyzed'.
We have endeavored to change this text as suggested except in cases referring to the interactions between EBV BORF2 and A3Bctd, since the results presented here together with our cryo-EM structure of the BORF2-A3Bctd complex (PMID 35476445) allow us to confidently say that L7 and L1 are the most critical determinants.
4) All microscopy figures need an A3 only panel (no RNR) to be able to judge relocalization.
Changed as suggested.
5) The matrix of labels above each IP blot is excessive since each lane only has one component that differs from the other lanes. A single label for each lane would make the plot easier to discern. These figures would also benefit from clearer labels including which virus each blot panel corresponds to (these could be along the left side of each blot; currently, the RNR gene name is provided, but this is a bit hard to find within the figure). Figures 2-4 would benefit from a label above each panel A indicating "L1" "L3" "L7".
Changed as suggested.
6) If the authors comment on pg9 ln 8 about intermediate relocalization effect, they should also mention 1C A3B L7G against BORF2.
Changed as suggested (page 8, lines 16-19).
7) Why is there no quantification of 6D relocalization? Could be supplemental if needed.
We have performed quantification of the relocalization phenotypes in Figures 2, 3 and 4 in order to allow direct comparison between WT and chimeric A3 enzymes in the presence of the same viral RNR (EBV BORF2 or KSHV ORF61). On the other hand, the images in Figure 6D are representative of the A3B/A relocalization phenotypes elicited by a larger panel of different viral RNRs. These representative images should be interpreted together with the co-IP and ssDNA deaminase activity assay data in Figures 6C and 6E, respectively.
8) Pg 6 ln 13 and Pg 8 ln2-4, ICP6 doesn't coIP w A3B; this should be clarified.
A similar concern was also raised by reviewer #2, and we agree that the ICP6 data present in the original version of this manuscript are not as easily interpretable compared to results with the RNRs from γ-herpesviruses such as EBV and KSHV. For the sake of clarity and cohesion, we decided to remove all of the HSV-1 ICP6 data from the revised version of our manuscript and focus on the A3B interactions with γ-herpesviruses.
9) Pg 8 ln21-23, the authors assume loss of function for A3G, but this swap could be functionally equivalent, but necessary for binding; it should be clarified that this is different than changing sequence and still binding.
Rephrased (page 9, lines 13-16).
10) Pg 9, ln 11, what is an anchor region?
We have removed the term “anchor region” and rephrased our text to more clearly describe the importance of L3 in KSHV ORF61 binding (page 10, lines 4-8).
11) Pg 9, ln 23, speculative – this might be explained by this 3 AA motif but it has not been tested.
Changed wording (page 10, lines 17-20).
12) Pg 10 ln 8, this doesn't show that this region is dispensable for binding, only that there is equivalent contribution or lack of contribution of function by the A and B loops, again assuming that the G loop is LOF.
Removed the phrase “indicating that this region may be dispensable for the interaction” (page 11, 1-2).
13) Pg 10 ln 10 – "can be explained by presence of bulky tryp" – this should be reworded to 'could or is likely caused by'.
Changed as suggested (page 11, lines 4-6).
14) Pg 11 ln 21, "can be explained by our cryo-EM" should be reworded to 'is supported by these contacts in cryo-EM'
Changed as suggested (page 12, lines 15-18).
15) Pg 13 ln 10 (and other places) dissociation rates are only part of affinity, Ka is equally important (pg 18 ln 8 also).
We agree and have revised our text account for this suggestion (page 13, lines 22-23; page 14, lines 1-2; page 22, lines 12-15).
16) Pg 14, ln 15 should be reworded to 'relocalize HUMAN cellular A3s'.
Changed as suggested (page 15, line 11).
17) Pg 16 Ln 16, this should be reworded as the data can't say it is completely dispensable without deletion of the loop.
Changed as suggested (page 21, lines 10-11).
18) New World monkeys have high activity of transposable elements of distinct types relative to catarrhines. It would be useful to mention that A3s restrict endogenous elements as well and how this might be a factor in the proposed evolutionary model.
This is a very interesting point that we plan to discuss in a future review. In addition, many future experiments will be needed to test the potential relationship between the birth of A3B and its potential impact on different classes of endogenous transposable elements.
19) Are the New World monkey viruses pathogenic in their native hosts? Perhaps not based on previous literature (reviewed in PMID: 11313011). This should be included in the discussion as it could certainly effect the evolutionary model for the birth/retention of A3 antagonism in these viruses.
While interesting, the observation that NWM herpesviruses do not cause disease in their native hosts is not unusual. In fact, most γ-herpesviruses (including human viruses like EBV and KSHV) have limited pathogenic potential when infecting their natural hosts (Fleckenstein B, Ensser A. Gammaherpesviruses of new world primates. Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis. 2007). Additionally, although the mentioned study (PMID: 11313011) reports asymptomatic infection of squirrel monkeys with SaHV-2, pathogenic infection/ oncogenic transformation have been reported following natural infection of marmosets with CalHV-3 (PMID: 11158621).
20) In previous papers on this topic, the lab has tested the effect of mutations on viral titers. While this may be beyond the scope of this paper, this would certainly elevate the paper and should be more clearly discussed.
As noted by the reviewer, we have previously demonstrated that A3B restricts EBV replication though a mutation-dependent mechanism and that this is counteracted by EBV BORF2 (PMID 30420783). While we completely agree that investigating the effect of A3B-catalyzed mutations on the titers of different γ-herpesviruses would be interesting, this would be technically challenging as we are currently not equipped to work with KSHV or any non-human primate herpesvirus.
21) What is the degree of sequence similarity among these and other RNRs? Is there any sense of what region of RNR binds A3s from the CryoEM structures and differences within these regions that might explain the functional differences?
We thank the reviewer for raising this important point. We have now included a new Figure 1 where we leverage the cryo-EM structure of the EBV BORF2-A3Bctd complex to make inferences about which regions of KSHV ORF61 may be involved in binding A3B/A. As described above, we also graft a short helical loop structure (HLS) from EBV BORF2 into the marmoset CalHV-3 RNR and showed that this small change enables the chimeric protein to bind to bind both human A3B and A3A (likely through L7), though not to the natural host marmoset A3A protein (Figure 7). Many additional interspecies chimeras could be constructed but we feel these are better suited for future studies (and specially to accompany future structural work in this area).
Reviewer #3 (Significance (Required)):
Previous work from the Harris lab showed that a subunit of the ribonucleotide reductase of some herpesviruses acts as an antagonist of several human APOBEC3s. Mechanistically, these viral protein block A3 inhibition by relocalizing nuclear A3s as well as inhibiting A3 deamination by binding and occluding the A3 active site. For Epstein-Barr virus, deletion of the antagonist (BORF2) results in a decrease in viral replication and accumulation of mutations likely introduced by host A3B that is no longer inhibited. However, deletion of the A3 antagonist from herpes simplex virus-1 (ICP6) had no effect on viral titers. Most recently, this group published a cryoEM structure of BORF2 in complex with the c-terminal half of A3B. This structure showed extensive contacts between BORF2 and two loops of A3B – L1 and L7.
The manuscript under review focuses on the previously suggested differences in the ability of different RNRs to bind A3A and A3B. This work provides an important contribution to this topic in defining specific regions of A3A and A3B and A3G that are necessary for viral RNRs to bind them. The variability in these interactions is surprising and likely testament to the impactful coevolution of herpesviruses and primate A3s. This manuscript will be of particular interest to virologists studying A3s or herpesviruses as well as evolutionary biologists interested in the rules of engagement between host restriction factors and viruses.
We thank you again for these thoughtful comments and for appreciating the overall significance of our work.https://doi.org/10.7554/eLife.83893.sa2
Article and author information
National Institute of Allergy and Infectious Diseases (R37-AI064046)
- Reuben S Harris
National Cancer Institute (P01-CA234228)
- Reuben S Harris
National Institute of Allergy and Infectious Diseases (F31-AI161910)
- Sofia N Moraes
National Institute of Allergy and Infectious Diseases (F32-AI147813)
- Jordan T Becker
National Institute of Allergy and Infectious Diseases (R56-AI150402)
- Nadine M Shaban
- Ashley A Auerbach
Howard Hughes Medical Institute (James H Gilliam Fellowships for Advanced Study program)
- Sofia N Moraes
- Reuben S Harris
National Institutes of Health (T32-AI83196)
- Nadine M Shaban
- Ashley A Auerbach
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We thank members of the Harris lab for thoughtful comments. These studies were supported in part by grants to RSH from the National Institute for Allergy and Infectious Diseases (NIAID) R37-AI064046 and the National Cancer Institute (NCI) P01-CA234228. Salary support for SNM was provided by NIAID F31-AI161910 and subsequently by a grant to the University of Minnesota (SNM and RSH) from the Howard Hughes Medical Institute through the James H Gilliam Fellowships for Advanced Study program. Salary support for JTB was provided by NIAID F32-AI147813. Salary support for NMS and AAA was provided in part by NIAID R56-AI150402 and NIH T32-AI83196, respectively. RSH is the Ewing Halsell President’s Council Distinguished Chair and an Investigator of the Howard Hughes Medical Institute. The authors have no competing interests to declare.
- Sara L Sawyer, University of Colorado Boulder, United States
- Jan E Carette, Stanford University School of Medicine, United States
- Preprint posted: April 4, 2022 (view preprint)
- Received: October 4, 2022
- Accepted: October 12, 2022
- Accepted Manuscript published: December 2, 2022 (version 1)
- Version of Record published: December 13, 2022 (version 2)
© 2022, Moraes et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
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