Rational attenuation of RNA viruses with zinc finger antiviral protein

Attenuation of a virulent virus is a proven approach for generating vaccines but can be unpredictable. For example, synonymous recoding of viral genomes can attenuate replication but sometimes results in pleiotropic effects that confound rational vaccine design. To enable specific, conditional attenuation of viruses, we examined target RNA features that enable zinc finger antiviral protein (ZAP) function. ZAP recognized CpG dinucleotides and targeted CpG-rich RNAs for depletion, but RNA features such as CpG numbers, spacing and surrounding nucleotide composition that enable specific modulation by ZAP were undefined. Using synonymously mutated HIV-1 genomes, we defined several sequence features that govern ZAP sensitivity and enable stable attenuation. We applied rules derived from experiments with HIV-1 to engineer a mutant enterovirus A71 genome whose attenuation was stable and strictly ZAP-dependent, both in cell culture and in mice. The conditionally attenuated enterovirus A71 mutant elicited neutralizing antibodies that were protective against wild-type enterovirus A71 infection and disease in mice. ZAP sensitivity can thus be readily applied for the rational design of conditionally attenuated viral vaccines.

T he zinc finger antiviral protein (ZAP) inhibits the replication of a broad range of RNA and DNA viruses 1-3 through the recognition of viral CpG-rich RNA 4 and presents opportunities for the design of attenuated viral vaccines. Live-attenuated viral vaccines offer advantages over other vaccine approaches because they express a complete repertoire of viral proteins and so present the widest range of antigenic determinants to induce durable cellular and humoral responses without adjuvants 5 . However, rational methods for the generation of attenuated viruses are few, and most attenuated vaccines have been empirically produced. One approach used for virus attenuation is recoding of nucleic acid sequences by synonymous mutagenesis. Initial reports using this method replaced codons or codon pairs with counterparts found only rarely in the human genome, a process termed 'deoptimization' [6][7][8][9][10] . However, deoptimization of a viral RNA genome can have pleiotropic effects on structure, stability and translation efficiency, conferring virus attenuation through multifactorial mechanisms that are not straightforward to predict 11 .
Attenuation by codon-pair deoptimization incidentally increases the frequency of two dinucleotides, CpG and UpA (TpA in DNA) 12 . CpG dinucleotides are severely underrepresented in vertebrate genomes, while TpA/UpA dinucleotides are underrepresented in organisms across the tree of life 13 . The underrepresentation of CpG in vertebrate genomes has created an opportunity for non-self RNA recognition that is exploited by ZAP proteins 4 . The ZAP N-terminal domain employs a highly selective binding pocket that can only accommodate a CpG dinucleotide in a single-stranded configuration 14,15 . While one ZAP molecule binds to one CpG dinucleotide, individual CpG dinucleotides have negligible effects on viral replication. Rather, it is the cumulative effect of multiple CpG dinucleotides that enables ZAP antiviral activity 4 . However, it is unknown how CpG dinucleotide numbers, juxtaposition and underlying sequence context affect ZAP recognition of viral RNA. Moreover, even though CpG dinucleotides confer ZAP sensitivity, introduction of CpG dinucleotides in an unguided manner can have pleiotropic effects on viral replication through ZAP-independent mechanisms 16 .
Viral genome recoding without consideration of the mechanism(s) of attenuation may produce viruses with reduced immunogenicity, which is clearly an undesirable property for any vaccine 17 . Since ZAP-RNA interactions may be immunostimulatory 18 , optimal recoding strategies would maximize ZAP binding and specify ZAP recognition as the attenuating mechanism. However, so far, delineation of sequence features that could be employed to achieve this goal has not been reported.
Using HIV-1 as a model system, we define how CpG dinucleotide number, spacing and surrounding sequence affect ZAP sensitivity. We then apply these parameters to design a mutant picornavirus genome with precise and stable modifications that function as an effective live-attenuated vaccine whose replication is specifically inhibited by ZAP in cell culture and in vivo.
synonymous substitution in the BstEII-ClaI bounded region (CG-1 to CG-23). While all mutants replicated indistinguishably from WT HIV-1 in ZAP-deficient cells, virus replication was progressively diminished in ZAP-expressing cells as the number of CpG dinucleotides was increased (Fig. 1c,d). Overall, CG-1 to CG-13 replicated well, CG-15 to CG-23 replicated poorly and CG-14 had an intermediate phenotype (Fig. 1c). The percentage of infected cells at 4 d post infection showed an obvious correlation between the number of introduced CpG dinucleotides and the extent of replication (Fig. 1d). Thus, individual CpG dinucleotides had an incremental impact, and approximately 15 CpG dinucleotides were required to profoundly inhibit HIV-1 replication.
Spacing and base composition between CpG dinucleotides and ZAP activity. We generated a second collection of HIV-1 mutants that each contained 15 CpG dinucleotides but differed in the spacing between each CpG dinucleotide. In ZAP-expressing cells, viruses that contained 15 CpG dinucleotides separated by a mean of 6 or 11 nucleotides replicated with near-WT kinetics. Conversely, viruses with CpG dinucleotides separated by 14 or 32 nucleotides were defective (Fig. 2a,b), while the effect of the 15 CpG dinucleotides was diminished if the spacing between them was further increased to a mean of 40 nucleotides.
To examine ZAP binding to these mutant viral sequences, we performed crosslinking immunoprecipitation assays coupled with RNA sequencing (CLIP-seq) using viruses with 15 CpG dinucleotides, separated by a mean of 11 or 32 nucleotides. We measured the frequency of CLIP-seq reads mapping to each nucleotide position in the BstEII-ClaI interval of the HIV-1 genome. While viral RNA containing no CpG dinucleotides in the BstEII-ClaI interval showed little ZAP binding, there was abundant ZAP binding when the CpG dinucleotides were positioned at a mean of 32 nucleotides apart (Fig. 2c). The cRISPR lesion results in the expression of truncated non-functional ZAP proteins indicated by asterisks. c, Replication of a collection of HIV-1 mutants containing between 0 and 23 cpG dinucleotides, as well as WT and cG-43 in ZAP-expressing or ZAP-deficient cells. At each day after initial infection, a small sample of cells was collected and the percentage of GFP-positive cells was measured by flow cytometry. d, Percentage of infected cells at day 4 post initial infection across all mutant viruses. Mean ± s.d. from 3 independent experiments; two-way ANOVA for the presence of ZAP (column factor) P < 0.0001, number of cpG (row factor) P < 0.0001. Šídák's multiple comparisons test was used to calculate adjusted P values between ZAP +/+ and ZAP −/− groups, comparisons of virus mutants with more than 3 cpG display P (adj) < 0.05.
However, ZAP binding to the modified sequence was minimal when CpG dinucleotides were positioned at a mean of 11 nucleotides apart (Fig. 2c). Thus, these data suggest that adequate spacing between CpG dinucleotides is important for ZAP recognition. Based on previous CLIP-seq experiments 4,13 and ZAP-RNA crystal structures 14,15 , ZAP specificity is determined solely by the target CpG dinucleotide and not flanking nucleotides. However, whether the overall sequence context in which CpG dinucleotides are present contributes to ZAP antiviral activity is unknown. We generated HIV-1 mutants that contained either 0 or 14 CpG dinucleotides (CG-0 and CG-14) and synonymously mutated the surrounding sequence in the BstEII-ClaI interval to contain the maximum possible number of adenine (A+), cytidine (C+), guanine (G+) or uridine (U+) nucleotides (Extended Data Fig. 2a). The CG-0 viruses with elevated A, U or G content (CG-0/A+, CG-0/ U+ and CG-0/G+) replicated with close to wildtype kinetics, while the cytidine-enriched virus (CG-0/C+) showed severe replication defects, independent of the presence of ZAP (Extended Data Fig.  2b,c). The CG-14/A+, CG-14/U+ and CG-14/G+ viruses replicated similarly to wild type in ZAP-deficient cells while the CG-14/ C+ virus exhibited a ZAP-independent defect similar to the CG-0/ C+ virus (Fig. 2d). Notably, while elevating G content (CG-14/G+) had little impact on virus replication, the CG-14/A+ and CG-14/ U+ viruses were severely attenuated, specifically in ZAP-expressing cells (Fig. 2d,e). Thus, increasing A or U content apparently increased the ability of CpG dinucleotides to impart ZAP sensitivity. We next generated 7 HIV-1 mutants, each containing 15 additional CpG dinucleotides, with the CpG-enriched sequences positioned at different locations across the env gene (Extended Data Fig. 3a). All these viruses replicated similarly to WT HIV-1 in ZAP-deficient cells, and 5/7 exhibited ZAP-dependent attenuation. The exceptions were two viruses with CpG-enriched segments located 3′ to env nucleotide positions 110 or 889 (CG-15(110) and CG-15(889), Extended Data Fig. 3b,c). Notably, the A and U frequencies in these two regions were reduced compared with other HIV-1 genome regions, and the mean spacing between CpG dinucleotides was the lowest among the mutants (Extended Data Fig.  3d). Strikingly, increasing the adenine frequency of CG-15(889) in the CpG-enriched interval to generate CG-15(889)/A+ increased ZAP-dependent attenuation such that CG-15(889)/A+ was specifically defective in ZAP-expressing cells (Extended Data Fig. 3d,e). We conclude that apparent position-dependent effects on ZAP sensitivity are probably mediated by surrounding nucleotide composition.
Selective pressure by ZAP can deplete CpG dinucleotides from viral genomes. The paucity of CpG dinucleotides in mammalian virus genomes may have been driven by ZAP selection 13,19 . The potential utility of reversing this property to generate ZAP-sensitive, live-attenuated vaccines depends on the stability of attenuating mutations. While codon-pair deoptimization has been reported to be stable during in vitro passage 20,21 , the stability of introduced CpG dinucleotides during viral passage under selective pressure by ZAP has not been assessed.
We performed long-term serial passage experiments with HIV-1 mutants containing 15 or 43 CpG dinucleotides (CG-15 and CG-43). Replication of CG-43 was severely inhibited in ZAP-expressing cells over the entire course of the experiment (43 d). Indeed, CG-43 never infected more than 1% of the cell population and no cytopathic effects typical of HIV-1 replication were observed ( Fig. 3a and Extended Data Fig. 4a). Conversely, the CG-15 virus initially replicated poorly in ZAP-expressing cells but was able to rapidly infect most of the cell population at later passages (Fig. 3b). Sequence analyses revealed a synonymous G-to-A transition at the wobble position of either a proline or a serine codon that caused the loss of a single CpG dinucleotide in each of the CG-15 experimental replicates, associated with increased replication (Extended Data Fig. 4b,c). Re-introducing these acquired point mutations into the parental CG-15 genome showed that the loss of a single CpG dinucleotide caused fitness recovery of CG-15 in ZAP-expressing cells (Fig. 3c). We conclude that attenuation of HIV-1 through introduction of a large number of CpG dinucleotides presents difficult-to-surmount genetic and stable attenuation barriers. Conversely, the presence of a number of CpG dinucleotides close to a threshold level enables emergence of ZAP-insensitive variants when selection pressure is applied.
Engineering enterovirus A71 to confer ZAP sensitivity. Members of the Picornaviridae are important human pathogens that cause morbidity 22 and most lack efficacious vaccines. One such picornavirus is enterovirus A71 (EV-A71) that causes hand, foot and mouth disease in young children, with occasional severe complications including acute flaccid paralysis, brainstem encephalitis and meningitis 23 . EV-A71 has a low frequency of CpG dinucleotides (Fig.  4a) and is therefore a good candidate for genetic recoding to confer ZAP sensitivity and generate a potential live-attenuated vaccine.
The EV-A71 genome encodes a single polyprotein (Fig. 4b) that is cleaved to generate structural (VP1, VP2, VP3 and VP4) and non-structural proteins (2A, 2B, 2C, 3A, 3C, VPg and the RNA-dependent RNA polymerase). To monitor EV-A71 replication, we generated a reporter virus encoding NanoLuc luciferase followed by a 2A cleavage site at the N terminus of the viral polyprotein, as previously described 24 . For recoding, we applied the CpG number, spacing and intervening mononucleotide content criteria determined above using HIV-1. We recoded a ~1 kb target region that spanned 2C, 3A, VPg and 3C coding sequences and was selected arbitrarily, other than the fact that it lacks any known proximal cis-acting RNA regulatory elements (Fig. 4b). In WT EV-A71, this region contains 32 CpG dinucleotides and 261 adenines (Fig.  4c). We changed the number and distribution of CpG dinucleotides, as well as the adenine content, generating three mutants: (1) EV-A71/A+ that contained a high frequency of A nucleotides without increasing the number of CpG dinucleotides; (2) EV-A71/ CG-48 with 16 additional CpG dinucleotides that combined with the existing 32 CpG dinucleotides, generating a segment with 48 CpG dinucleotides at a mean of 19 nucleotides apart but retaining WT mononucleotide composition; and (3) EV-A71/CG-48/A+ that had the 16 additional CpG dinucleotides positioned as above but in the A-rich sequence context (Fig. 4c). All viruses replicated well in ZAP-deficient cells ( Fig. 4d-f). While no replication defects were observed in the WT EV-71, EV-A71/A+ or EV-A71/CG-48 mutants, the EV-A71/CG-48/A+ mutant was specifically attenuated in ZAP-expressing cells ( Fig. 4d-f).
When the EV-A71/CG-48/A+ mutant was repeatedly passaged in ZAP-expressing cells, luciferase activity progressively decreased with each passage and was ultimately below the detection limit ( Fig.  4g). Conversely, no replication defect was evident in ZAP-deficient cells, and sequence analyses revealed no reversion mutations acquired during passage. While viral RNA was abundant in all EV-A71/CG-48/A+ replicates in ZAP-deficient cells, RNA levels were below the detection limit in ZAP +/+ cells (Extended Data Fig.  5a,b). Moreover, when we followed virus replication for 4 days using NanoLuc assays, determination of infectious virus yield (TCID 50 ) and viral RNA quantification, all three measurements of EV-A71/ CG-48/A+ mutant replication were dramatically reduced in ZAP +/+ cells compared with ZAP-deficient cells (Extended Data Fig. 5c-e). Thus, the observed replication deficits reflected bona fide effects of ZAP and not reporter gene instability. Moreover, the CpG and A enrichment was stable and EV-A71/CG-48/A+ could not escape ZAP under these conditions. Thus, the principles governing ZAP sensitivity identified using HIV-1 could be applied to an unrelated RNA virus, leading to stable, ZAP-dependent attenuation.
Recoded EV-A71 elicits protective immunity in mice. We collected plasma from ZAP +/+ mice 5 weeks after previous inoculation with mEV-A71/CG-48/A+ at 1 d or 5 d of age (Fig. 5e). While plasma from mock-inoculated mice did not neutralize EV-A71, plasma from mice inoculated with mEV-A71/CG-48/A+ neutralized EV-A71 infection, with 50% neutralizing titres (NT 50 ) ranging from 895 to 10,602 in mice infected at 1 d of age (median NT 50 = 2,957) and titres ranging from 627 to 1,514 in mice infected at 5 d of age (median NT 50 = 1,162) ( Fig. 5f and Extended Data Fig. 8a). Next, we aimed to determine whether these neutralizing antibodies were protective in vivo. Since productive infection of mEV-A71 is age-sensitive in mice 28 , we performed passive protection experiments in which the neonatal offspring of females that were previously mock-inoculated or inoculated with mEV-A71/ CG-48/A+ were challenged with WT mEV-A71 (Fig. 5e). In this type of experiment, suckling pups acquire antibodies via maternal milk 26 . Pups from females previously inoculated with mEV-A71/ CG-48/A+ at 1 d or 5 d of age (Fig. 5e) showed reduced disease (median clinical score at day 20 of 0.52 and 0.24 in 1-day-old and 5-day-old infected mice, respectively) and increased survival compared with pups from mock-treated females (median clinical score at day 20 of 3.14 and 2.86 in 1-day-old and 5-day-old infected mice, respectively) ( Fig. 5g and Extended Data Fig. 8b). We conclude that ZAP-attenuated mEV-A71/CG-48/A+ replication in mice elicits antibodies that are passively transferred and protective against mEV-A71 disease in the offspring of inoculated females.

Discussion
The delineation of sequence features that affect sensitivity to ZAP in HIV-1 (numbers of CpG, spacing and context) enabled us to develop design rules that we applied to engineer a picornavirus mutant that is strongly attenuated in a strictly conditional manner. The close spacing between each CpG dinucleotide affecting ZAP sensitivity and ZAP binding in CLIP-seq suggests that ZAP molecules binding to adjacent CpG dinucleotides may compete with each other, consistent with modelling studies indicating that ZAP binds RNA sequences of ~13 nucleotides 29 . Closely spaced CpG dinucleotides may also promote RNA secondary structure that might inhibit ZAP access 30 . Conversely, wide CpG spacing conferred reduced sensitivity. Interactions between ZAP and TRIM25 13 and between ZAP and KHNYN 31 , two known co-factors of ZAP, appear to be mediated by protein-protein contacts; thus, it is possible that ZAP molecules and co-factors bound to adjacent CpG dinucleotides may coalesce, with close spacing facilitating assembly of active ZAP:TRIM25:KHNYN complexes.  characteristic pathology developed in infected mice following meV-A71 infection, including one-limb and two-limb paralysis. c, Probability of ZAP +/+ and ZAP −/− mouse survival (%) following infection with meV-A71 WT or meV-A71/cG-48/A+ (n = 11-36 mice per group; P value calculated using Mantel-cox test). d, One-day-old mice (n = 4-6 per group) were infected with indicated virus; 6-days-post-infection mice were sacrificed and muscles from both posterior limbs were collected, homogenized and total RNA was extracted. eV-A71-specific RNA was quantified by qPcR. Dashed line indicates limit of qPcR detection. e, Schematic representation of experimental design. ZAP +/+ Ifnar −/− female mice previously inoculated with meV-A71/cG-48/A+ (or mock-infected females) were mated with ZAP +/+ Ifnar −/− male mice. Resulting offspring was challenged at 1 d of age with meV-A71 WT. f, Neutralizing activity in plasma from mice after meV-A71/cG-48/A+ infection (infection at 1 d of age), blood collection at 5 weeks, or mock-infected mice evaluated using eV-A71 NanoLuc luciferase reporter virus. 293T cells were incubated with the antibody:virus mixture for 48 h and luciferase activity was measured. g, clinical score and survival probability following meV-A71 (WT) infection of the offspring of ZAP +/+ /IFNAR −/− females previously inoculated with meV-A71/cG-48/A+ (n = 25 pups from 4 different females) or previously mock-infected (n = 22 pups from 4 different females) at 1 d of age. clinical score and survival were assessed daily until weaning. Statistical significance was inferred by two-way ANOVA and Mantel-cox tests.
Studies of RNA-binding protein specificity typically focus on the recognition of particular RNA sequences. Although many RNA-binding proteins recognize specific sequence motifs, contextual features, such as flanking nucleotide composition, can be crucial for determining target specificity 32 . Increasing adenine or uridine content in viral genomes may increase sensitivity to RNAses, such as RNAse L that cleaves UpA and UpU dinucleotides in viral RNA, and we note that our A-or U-enriched viral genomes contain greater numbers of UpA dinucleotides 33,34 . Nevertheless, A/U-enriched viruses replicated identically to wildtype HIV-1 in ZAP-deficient cells, indicating that the defects imposed by A/U enrichment are ZAP specific. An obvious effect of A or U enrichment would be to reduce stable secondary RNA structure, perhaps increasing CpG dinucleotide accessibility to ZAP.
All approaches to viral attenuation must balance reduced pathogenesis versus reduced antigen levels that accompany impaired viral genome expression and replication. In principle, programed attenuation of viruses based on ZAP sensitivity might be adjustable through variation in CpG number and accessibility. Because RNA features conferring ZAP sensitivity in HIV-1 were readily transferrable to a very different virus (EV-A71), these approaches may be generally applicable, and it is possible that nearly any virus that exhibits CpG depletion could be a suitable target for this recoding approach. Importantly, no loss-of-function mutations in ZAP have been identified so far in humans, but further investigation would be required to evaluate whether existing genetic variation in ZAP might render some individuals more susceptible to CpG-enriched viruses and whether the stability of the introduced mutations that we observed for HIV-1 and EV-A71 in cell culture and in mice would be generalizable to CpG-enriched virus-vaccinated humans. Attenuation through engineered ZAP sensitivity might be ineffective for viruses with alternative mechanisms of ZAP evasion, such as targeting ZAP or co-factors for depletion, although no such viruses are currently known. Notably, strictly ZAP-dependent attenuation allows for the cultivation of high-titre, live-vaccine stocks in ZAP-deficient cells. Additional advantages may stem from the reported observation that RNA recognition by ZAP is immunostimulatory 18 . Indeed, infection of mice with CpG-enriched influenza A viruses elicited immune responses that were disproportionate to the level of virus replication 35 . ZAP-independent attenuation through unguided deoptimization may forego these benefits. We also note that the principles described herein may also prove useful in the engineering of nucleic acid-based gene delivery vectors. Indeed, CpG dinucleotides depletion from the DNA of gene delivery vectors was reported to improve performance 36,37 .
In summary, our results identify sequence features that are important for recognition of foreign RNA by ZAP and thereby enable highly specific rational attenuation of two different RNA viruses. Our findings establish design principles for engineering CpG-enriched viruses that are conditionally attenuated and can elicit a protective immune response in mice, thereby paving the way to the rational design of live-attenuated viruses with vaccine potential. Sequence design and plasmid construction. An HIV-1 proviral plasmid containing the enhanced green fluorescent protein (EGFP) gene in the Nef position (NHG, GenBank: MF944225.1) was engineered to contain unique BstEII and ClaI restriction sites at nucleotide positions 6325 and 6771, respectively, within the env gene. DNA sequences comprising the coding region between the BstEII and ClaI restriction sites were designed to modulate the number and location of introduced CpG dinucleotides as well as the background mononucleotide frequency, without changing the encoded amino acids. For mutants in which the number of CpG dinucleotides was changed, the CpG dinucleotides were introduced at the codon boundaries by replacing the wobble position of the 5′ codon by a cytidine, or by substituting the wobble position of alanine, threonine, proline and serine codons with a guanine. The sequences surrounding each CpG dinucleotide was retained as in the HIV-1 NHG wildtype sequence. For mutants with variable spacing between each CpG dinucleotide and in mutants with different locations across the Env gene, a similar approach was adopted in which the wobble position of codons was modified to introduce a CpG at codon boundaries or within codons. Similarly, for modified wobble positions, the surrounding sequences were maintained as they appear in the wildtype sequences, with an exception: in some spacing mutants, four serine codons were substituted (AGT/AGC-->TCG). Finally, mutants with modified mononucleotide composition were designed by maintaining the position of the 14 CpG dinucleotides in the CG-14 virus and replacing all other codons with codons that contained the desired nucleotide. For example, in A+ mutants, leucine codons (CUU) were replaced with adenine-containing leucine codons (CUA), arginine codons (AGG) with adenine-containing codons (AGA) and so on. In all cases, no additional CpG dinucleotides were introduced. All sequences were designed with in-house built scripts and checked for inadvertent introduction of splice sites using MaxEntScan 38 . Synthetic DNA sequences encoding the modified sequences were purchased (Twist Bioscience) inserted into the HIV-1 NHG BstEII and ClaI modified proviral plasmid using standard cloning procedures.

Cells and animals. Human embryonic kidney (HEK) 293T ZAP
Plasmids encoding the enterovirus A71 strain 41 24 were used as a basis for the construction of the EV-A71 mutants. DNA sequences encoding the enteroviral polyprotein region between D1270 and R1586 were designed to modulate the number and location of CpG dinucleotides as well as the surrounding adenine content, without changing the amino acid sequence, as described above. Synthetic DNA sequences encoding the modified sequences were purchased (Twist Bioscience) and inserted into the EV-A71 genome plasmid using the BstEII and SacII restriction sites. A reporter EV-A71 encoding NanoLuc luciferase was generated as previously described 24 by inserting the NanoLuc gene followed by a 2A cleavage site at the N terminus of the EVA-71 polyprotein. Mouse-adapted versions of EV-A71 wild type and EV-A71/CG-48/A+ were generated by inserting the mouse-adaptive substitutions described previously 25 . These mutations are K149I in VP2, plus Q145E and K244E in VP1. Additionally, to improve the replication of these viruses in human cell lines, the previously described substitution (H37K in VP1) 27 was also introduced.

Virus production.
To produce HIV-1 mutant and wildtype virus stocks, HEK293T ZAP −/− TRIM25 −/− cells were transfected with HIV-1 NHG proviral plasmids along with a plasmid encoding the vesicular stomatitis virus glycoprotein (VSV-G). The next day, cell culture media were replaced and at 48 h post-transfection, supernatants were collected, clarified by centrifugation (10 min, 2,000 × g) and filtered through a 0.22 µm filter. Collected viruses were concentrated using Lenti-X concentrator (Clontech) according to the manufacturer's guidelines and resuspended in serum-free DMEM.
EV-A71 wildtype and mutant viruses were generated as described previously 24 . Briefly, viral plasmids were linearized with MluI restriction enzyme and column purified. Linearized DNA was then used to generate viral RNA using the T7 RiboMAX Express large-scale RNA production system according to the manufacturer's guidelines. Viral RNA was then transfected in ZAP-deficient RD cells using the TransIT-mRNA transfection kit. After overnight incubation, media were replaced and cells were monitored for cytopathic effect. When cytopathic effect was observed in ~80% of cells, supernatants were collected and filtered through a 0.1 µm filter. Virus stocks were passaged once in ZAP-deficient RD cells. All virus stocks were stored at −80 °C before use.

HIV-1 replication assays.
For spreading infections, 1.5 × 10 5 MT4 cells were infected with 400 infectious units of VSV-G pseudotyped HIV-1 NHG in a total of 2 ml of complete RPMI. Each day after infection, cells were resuspended, 100 µl of cell suspension was collected and fixed in 4% paraformaldehyde, and cultures were replenished with 100 µl of fresh RPMI. The percentage of GFP-positive cells was determined using flow cytometry and calculated using FlowJo. For the long-term virus passage experiments, 7.5 × 10 5 MT4 cells were infected with 2,000 infectious units of the CG-43 or CG-15 HIV-1 NHG mutants. Every 2 d, cells were resuspended, 100 µl of the cell suspension was fixed in 4% paraformaldehyde and the percentage of GFP-positive cells was measured using a flow cytometer. When the percentage of GFP-positive cells was greater than 85%, supernatants were collected, filtered through a 0.22 µm filter and used as inoculum to infect fresh MT4 cells. Viral RNA was isolated from an aliquot of the passaged supernatant using TRIzol, reverse transcribed using the SuperScript III first-strand synthesis system, and an env fragment was amplified using the following primers: 5′-ACAGAAAAATTGTGGGTCACCGTCTATTATGGG-3′ and 5′-GCTGGTAGTATCATTATCGATTGGTATTATATCAAG-3′. Mutations identified in revertant viruses (S115 G6565A, P118 G6574A) were then introduced into the HIV-1 NHG CG-15 construct by site-directed mutagenesis. Virus stocks were generated as described above and their replication assessed in a spreading infection assay.
EVA-71 replication assays. HeLa cells were infected at a multiplicity of infection (MOI) of 0.02 for 1 h at 37 °C. Cells were then washed twice in PBS and incubated in complete DMEM at 37 °C. At the indicated timepoints, 100 µl of the culture supernatant was collected and incubated with 25 µl of 5x concentrated passive lysis buffer (Promega) at room temperature for 5 min. NanoLuc luciferase activity was measured using the Nano-Glo luciferase assay system (Promega) according to the manufacturer's guidelines. For long-term virus passage experiments, cells were infected as above and at 4 d post infection, supernatants were collected, clarified by centrifugation (10 min, 2,000 × g) and filtered through a 0.22 µm filter. Collected virus was diluted and used to re-infect cells at MOI = 0.02. Median tissue culture infectious doses (TCID 50 ) were determined using a cytopathic effect readout and calculated as previously described 39 using RD ZAP −/− target cells.
Immunoblotting. Cells were lysed in NuPAGE LDS sample buffer (Invitrogen) supplemented with β-mercaptoethanol. Samples were then heated at 72 °C for 20 min and sonicated for 15 s. Protein samples were resolved onto NuPAGE 4-12%, Bis-Tris protein gels (Invitrogen), transferred to nitrocellulose membranes and blocked with Intercept blocking buffer (Li-Cor) and incubated with the following antibodies: anti-ZC3HAV1 (rabbit polyclonal antibody, 16820-1-AP, Proteintech) was used at 1:5,000 dilution in PBS supplemented with Tween20 in human MT4 cell line samples, anti-ZC3HAV1 (rabbit polyclonal antibody, abx124715, Abbexa) was used at 1:300 dilution in 5% milk in PBS-Tween20 in mouse peripheral blood mononuclear cell samples and anti-α-tubulin (mouse monoclonal antibody, T5168, Sigma). After overnight incubation at 4 °C, membranes were washed in PBS-Tween20, blotted with horseradish peroxidase-conjugated secondary antibodies and developed using a C-Digit chemiluminescent western blot scanner.

CLIP-seq.
All CLIP-seq experiments were performed as described previously 13 with the following modifications. HEK293T ZAP −/− TRIM25 −/− cells were transfected with a proviral plasmid and a plasmid encoding the long isoform of ZAP (ZAP-L) and three consecutive C-terminal hemagglutinin (HA) epitope tags. The next day, media were replaced and 4-thiouridine was added to the culture for 16 h. Cells were then washed with cold PBS and exposed to UV radiation. ZAP:RNA complexes were isolated by immunoprecipitation with an anti-HA antibody and RNA was ligated to a fluorescently labelled 3′ adapter. RNA was isolated and ligated to a 5′ adapter and reverse transcribed using the SuperScript IV first-strand synthesis system (Invitrogen). The resulting complementary DNA library was amplified using Illumina primers and sequenced using a NovaSeq sequencer (Rockefeller Genomics Resource Center). Reads were processed as described previously 4 and aligned against the HIV-1 NHG genome as indicated.
Mouse experiments and generation of ZAP-knockout mice. All mice used in this study were derived from the C57BL/6 line or the C57BL/6J Ifnar1 −/− knockout line (MMRRC, 32045) 40 . Mice of both sexes were used, except for the passive protective experiments in which females were specifically used (to generate immunized dams). All animal experiments were conducted according to The Rockefeller University Institutional Animal Care and Use Committee.
Mouse infections with mEV71. To assess replication and pathogenicity of mEV-A71 and mutants thereof, 1-day-old suckling mice were infected by intraperitoneal injection with 1 × 10 5 TCID 50 of mouse-adapted mEV-A71 (wildtype) or mEV-A71/CG-48/A+. Pups were monitored daily for symptoms for a total period of 20 d. A clinical score was measured as described previously 26 with the following modifications: 0, healthy; 1, weak/lethargic; 2, one-limb paralysis; 3, two-limb paralysis; 4, dead/moribund/euthanized. When two-limb paralysis was observed for a period of 48 h without recovery, affected mice were humanely euthanized. For the measurement of viral RNA, mice were euthanized at 6 d post infection. Skeletal muscle from rear limbs were collected and stored in RNAlater solution (Thermo) before processing. Organs were thawed and homogenized in TRizol LS reagent according to the manufacturer's guidelines. Isolated RNA was reverse transcribed using the SuperScript III first-strand synthesis system (Invitrogen) and viral RNA molecules were quantified by quantitative RT-PCR using TaqMan gene expression master mix and the following oligonucleotides: probe EV-A71: 5′-6FAM-ATTCCAAAAGAAAGCACTATCCAGTCAGC-MGBNFQ-3′, forward primer: 5′-GAACCTCGTCTGGGAAGATAGCTCCC-3′ and reverse primer: 5′-TCGCCGGGCTCAGAGTGGCCT-3′.
For the passive protection experiments, female ZAP +/+ Ifnar1 −/− mice that were previously infected with mEV-A71/CG-48/A+ virus as well as mock-treated females at 6-weeks of age were mated with naïve Ifnar −/− males. The resulting offspring were infected with mEV-A71 wild type via the intraperitoneal route as described above. Infected mice were monitored daily until weaning age as described above.
EV-A71 neutralizing antibody assays. Blood samples from mice inoculated with mEV71/CG-48/A+ were collected at 4 and 6 weeks after infection. Plasma was heat-inactivated as described previously 41 , serially diluted and incubated with EV-A71 NanoLuc reporter virus for 1 h at 37 °C. Viruses were then used to infect HEK293T cells and incubated for a further 48 h at 37 °C. Infected cells were then lysed with passive lysis buffer (Promega) and nano luciferase activity was measured as described above. NT 50 titres were calculated in GraphPad Prism using non-linear regression (least squares regression without weighting).
Statistical analysis. All statistical analyses were performed using GraphPad Prism 9. Spreading infection data were plotted as mean ± s.d. For comparison between virus mutants and presence of ZAP, two-way analysis of variance (ANOVA) was used with Šídák's multiple comparisons test. Quantified viral RNA and luciferase activity were plotted as mean ± s.d. For the plasma neutralization data, both mean and SEM were plotted. We chose the indicated sample size for our animal experiments on the basis of our previous experiments using C57BL/6 mice in infection models 41 . To assess statistical significance in the clinical scores of infected mice, we performed two-way ANOVA, while differences in probability of survival were assessed using the Mantel-Cox test. Details of statistical tests, sample sizes as well as P values are described for each experiment in the respective figure legends.
Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
The data that support the findings of this study are available in the accompanying Source Data files. The NHG HIV-1 genome sequence used in this study can be accessed through the NCBI nucleotide database using the accession code MF944225.1. Unprocessed raw data from CLIP-Seq experiments can be accessed through the NCBI Gene Expression Omnibus database using the accession code GSE208611.

Code availability
Code used to map and calculate read counts from CLIP-seq experiments was obtained from http://hannonlab.cshl.edu/fastx_toolkit/ and can be accessed from GitHub under https://github.com/agordon/fastx_toolkit.

March 2021
Corresponding author(s): Paul D. Bieniasz Last updated by author(s): Jul 27, 2022 Reporting Summary Nature Portfolio wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Portfolio policies, see our Editorial Policies and the Editorial Policy Checklist.

Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.

n/a Confirmed
The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted

Software and code
Policy information about availability of computer code Data collection CLIP-Seq data were processed and analyzed using python scripts as part of the FASTX-Toolkit version-0.0.14 (available here: Code used to map and calculate read counts from CLIP-Seq experiments is available from http://hannonlab.cshl.edu/fastx_toolkit/ and can be cloned from GitHub under https://github.com/agordon/fastx_toolkit).

Data analysis
Flow cytometry data were analyzed using FlowJo version 10.8.0. All other data were plotted and analyzed using GraphPad Prism version 9.2.0. Statistical test were performed using GraphPad Prism version 9.2.0. Analysis of potential splicing sites in recoded sequences was performed using MaxEntScan (available here: http://hollywood.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html) For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Portfolio guidelines for submitting code & software for further information.

March 2021
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy The data that support the findings of this study are available in the Source Data files. Sequencing data resulting from CLIP-Seq experiments have been deposited in the NCBI GEO database and can be accessed using the accession code GSE208611. The NHG HIV-1 genome we used in this study can be accessed through the NCBI Nucleotide database using the accession code MF944225.1.

Human research participants
Policy information about studies involving human research participants and Sex and Gender in Research.
Reporting on sex and gender Note that full information on the approval of the study protocol must also be provided in the manuscript.

Field-specific reporting
Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative. Data exclusions No data was excluded from this study with the exception of some CLIP experiments with read counts that were too low to be reliable.

Replication
All experiments presented in this manuscript were reliably replicated, at least 3 times, and presented equivalent results. CLIP experiments were performed twice, since these are inherently complex experiments with frequent reagent deterioration. Both experiments yielded remarkably similar results with very high read counts.
Randomization All mice were infected at the same age and with the same infectious dose. Littermates of both sexes were included in the same experimental groups. Mice litters were randomly chosen to be infected with wildtype or mutant viruses as they became available from ongoing matings. Female mice that survived infection were then paired with non-exposed males; their offspring was challenged with wiltype viruses as mice litters become available without deliberate selection. All other experiments that did not involve mice were not subject to randomization since experimental units measurements were performed by machines and not subject to operator's bias.

Blinding
No blinding was used in this study since most of the quantitative data presented were measured by a machine. In infection experiments in mice that required the attribution of a previously published clinical score, we based scores on the observation of clear and obvious symptoms including death of the animal, the complete paralysis of 1 or 2 limbs, and hunched position adopted by some mice. All infections in mice resulted in a binary survival score (dead or alive) that could not be subject to interpretation by the operator.
Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.