Capsid Killers: How VP1 and VP2 Throttle AAV Genome Packaging

1. Introduction

The AAV capsid is a ~25 nm icosahedral shell composed of 60 subunits derived from three derivative proteins (VP1, VP2, and VP3) in an approximate 1:1:10 molar ratio (Penaud-Budloo et al. 2018). All three share the VP3 core, which forms the shell and binds the receptor; VP2 adds an N-terminal extension, and VP1 adds a longer one that carries a phospholipase A2 (PLA2) domain essential for endosomal escape and basic-region nuclear-localization signals (NLS) required for nuclear import (Bleker et al. 2005, Girod et al. 2002). In the assembled particle, these N-terminal extensions are sequestered inside the capsid and externalized only upon endosomal pH activation, threading out through the fivefold pore during cell entry (Bennett et al. 2017).

The companion paper (Davis 2026) found a ceiling effect in productive packaging during stochastic genome loading. Here we ask whether the capsid’s architecture imposes a second, structural constraint upstream of loading. If the VP1/VP2 N-terminal peptides occupy the fivefold channels through which DNA must pass, then a capsid’s packaging-competent channels are limited to those pentons assembled entirely from VP3. VP1 and VP2 are termed, in a narrow manufacturing sense, “capsid killers”, not because they damage transduction (they are, of course, essential for infectivity), but because, by occupying channels, they can prevent productive one-genome-per-capsid packaging and thereby increase the fractions of empty and defective-intermediate (DI) particles.

It is important to distinguish the model’s prediction from a naive interpretation of the obstruction hypothesis. At the canonical VP ratio, channel obstruction has negligible effect on whether a capsid can be packaged and does not raise the stochastic ceiling for “authentic” single-genome capsids. Its principal signature is threshold behavior: when VP1 and VP2 are over-incorporated, as can occur in recombinant systems, the open-channel fraction declines nonlinearly, and empty capsids become predominant. The key result is therefore not a universal reduction in packaging capacity but a stoichiometry-dependent transition. At fixed genome supply, obstruction also produces a counterintuitive tradeoff: it reduces multi-occupancy defectives while increasing empty capsids rather than compounding the defect. Accompanying figures illustrate this behavior.

1.1. The Fivefold Pore Is the Packaging Portal

Genome packaging in AAV, as in related parvoviruses, proceeds via Rep-motor-driven translocation of single-stranded DNA through a pore at a fivefold vertex into a preformed capsid (Bleker et al. 2006, Bleker et al. 2005, King et al. 2001). Mutations lining the AAV2 fivefold pore impair packaging through steric and Rep-interaction effects (Bleker et al. 2006, Bleker et al. 2005). A similar result in minute virus of mice (MVM), where a pore-occluding mutation abolishes packaging, confirms the fivefold channel as the portal (Plevka et al. 2011). The capsid has twelve such channels.

1.2. VP1, VP2, VP3: Shared Core, Distinct N-Termini

The three VP proteins arise from a single cap ORF via alternative splicing and start-codon usage; all share the VP3 core, with VP2 carrying ~65 additional N-terminal residues and VP1 ~135 more beyond VP2, including the PLA2 domain and the basic regions BR1, BR2, and BR3, which function in nuclear localization and import (Girod et al. 2002, Grieger et al. 2007). VP3 has no N-terminal extension and thus contributes no intraluminal peptide. Only the VP3 core is required for capsid assembly; particles can be assembled from VP3 alone, and VP-reduced particles can package the genome (Grieger et al. 2007, Warrington et al. 2004).

1.3. The N-Termini Are Internal and Must Reach the Interior Through the Pore

Immunological, proteolytic, and cryo-EM studies predominantly place the VP1/VP2 N-termini inside the native capsid, externalizing only upon acidification during entry (Bennett et al. 2017, Kronenberg et al. 2005). This paper assumes that the most parsimonious structural path for an N-terminal peptide in the completed capsid is through the fivefold pore in the penton where its subunit sits. Notably, recent structural work indicates that the fivefold pore is largely empty before packaging and becomes occupied by capsid-protein N-terminal density only after packaging (Kaelber et al. 2025). This timing is discussed in Section 1.5 because while it is the main objection to the hypothesis, it remains an unsettled question.

1.4. VP Stoichiometry Is Stochastic and Variable

Native mass spectrometry shows that AAV capsids are not uniform: they assemble from 60 random draws from the expressed VP pool, yielding a broad distribution of per-capsid VP compositions centered on the 5:5:50 mean ,(Wörner et al. 2021). This stochastic assembly model underpins my “channel availability” calculation. Critically, the VP1:VP2:VP3 ratio is not fixed: it varies by serotype, platform, and process. Recombinant systems under heterologous promoters can also deviate substantially from 1:1:10, sometimes with elevated VP1/VP2 (Wörner, 2021 #23). These variations, as will be shown, can have significant consequences for packaging.

1.5. The Channel-Obstruction Hypothesis

The proposed model holds that a penton containing one or more VP1 or VP2 subunits is non-permissive for ssDNA insertion because the intraluminal N-terminal peptide of the corresponding subunit occludes the fivefold channel. In this model, only pentons composed entirely of VP3 present packaging-competent channels. This steric hindrance argument is consistent with reported AAV fivefold-channel constrictions of 9–20 Å (Bleker et al. 2006, Bleker et al. 2005, Kronenberg et al. 2005). A translocating single strand of DNA has an effective transverse dimension of 11–13 Å, about half the double-stranded dimensions reported by Mandelkern et al. (Mandelkern et al. 1981), whereas an extended, denatured, unhydrated polypeptide chain exceeds 4–6 Å (Buscaglia et al. 2006). Given these dimensions, a resident peptide and a translocating DNA strand are unlikely to simultaneously occupy the same constriction. It would seem, therefore, that even a single N-terminal peptide chain resident in the channel would obstruct ssDNA passage during packaging. While plausible, this interpretation has not been established with certainty primarily because the capsid channel is conformationally dynamic, and the identity of the resident pore density visualized by cryo-EM remains unresolved.

A recent cryo-electron microscopy study of AAV5 could, upon substantiation, require an important refinement of the model. Gliwa and colleagues (Gliwa et al. 2025) resolved a rod-like density within the fivefold channel and most plausibly assigned it to the VP3 N-terminus. They also raised the possibility that the twelve pores are not equivalent: most may contain VP3 N-terminal density, whereas a minority of specialized pores may mediate genome traffic. However, the authors explicitly note that this specialized-pore interpretation remains speculative (Gliwa et al. 2025). These observations are relevant because they broaden the structural interpretation of channel occupancy. However, they do not resolve the central question addressed here: whether the longer VP1/VP2 N-terminal extensions, when present in a packaging channel, impose a greater and more persistent obstruction to ssDNA entry than the shorter VP3 N-terminus. Two qualifications therefore follow.

First, if the shared VP3-core N-terminus contributes density within the fivefold channel, an all-VP3 penton should not be assumed to be necessarily empty. Rather, the relevant distinction is the magnitude, persistence, and functional consequence of channel occupancy. In that framework, the longer VP1 and VP2 extensions are still expected to pose a greater steric barrier to ssDNA passage than the shorter VP3 N-terminus. Second, pore-to-pore heterogeneity may provide an additional mechanism by which a subset of channels remains competent for genome traffic, without requiring all pores to be structurally equivalent. Because the new evidence comes from AAV5, whereas several pore-dimension estimates cited here derive from AAV2, the cross-serotype inference should be regarded as provisional. These qualifications refine the structural interpretation but do not weaken the model’s central quantitative prediction: VP1/VP2 over-incorporation should reduce the fraction of packaging-competent capsids in a threshold-dependent manner, thereby increasing the empty-capsid fraction; a prediction testable by VP-ratio titration, independent of a complete structural resolution of the packaging intermediate.

Most importantly, this model differs from the prevailing structural interpretation, which holds that the fivefold pore remains unobstructed during packaging and that N-terminal occupancy occurs only after genome encapsidation. The timing of N-terminal occupancy during active packaging has not yet been directly observed: mature-particle structures describe the post-packaging state, whereas packaging-intermediate structures that could resolve the timing question are absent. Thus, if the N-terminal peptide of a VP1 or VP2 subunit is present in or near its penton channel when Rep attempts to load that channel, obstruction should occur. The model therefore predicts that capsids captured during packaging will show N-terminal density in channels associated with VP1/VP2-containing pentons, thereby partitioning channels into competent all-VP3 and obstructed VP1/VP2-containing classes. It further predicts that experimentally increasing the VP1/VP2 fraction will increase the empty-capsid fraction in the threshold-dependent manner derived below, independently of genome supply. Conversely, a demonstration that VP1/VP2 N-termini are consistently excluded or retracted from the pore throughout packaging would refute the proposed mechanism and provide important insight into encapsidation and manufacturing control.

2. Materials and Methods

2.1. The Channel-Availability Model

The AAV capsid has twelve fivefold channels, one per vertex, each formed by a pentamer of five subunits. Under the stochastic-assembly model of VP composition (Wörner, 2021 #23), each of the 60 subunits is an independent draw, with VP3 occurring with probability equal to the VP3 mole fraction f_VP3. The twelve pentons occupy spatially distinct sets of five positions and are therefore independent. A penton is packaging-competent (“open”) only when all five of its subunits are VP3. The probability that a given penton is open is P(open) = f_VP3⁵, and the number of open channels per capsid is binomially distributed as B(12, f_VP3⁵). To couple channel availability to genome loading, each open channel is modeled as independently loading a DNA molecule with probability π, set by genome supply and shared across the population, so that inserts per capsid follow Binomial(c, π) with bulk MOE = E[c]·π. Both models were evaluated across the full ranges of f_VP3 and π; the binomial derivation is given in the Appendix.

2.2. Model Development and Computational Methods

The channel-obstruction hypothesis and its quantitative consequences were developed by the author, building on the established stochastic-assembly model of VP composition. During manuscript preparation, the generative AI assistant Claude Opus 4.8 (Anthropic) was used under the author’s direction and verification to: (i) express the channel-availability and coupling models in compact symbolic form; (ii) evaluate the binomial and coupled distributions across the full range of VP3 mole fraction and per-channel loading probability; and (iii) compute and plot the resulting parameter-space behavior. Notably, this analysis made explicit the sharp stoichiometric threshold, or “knee,” near a VP3 mole fraction of 0.75, which had not been fully resolved in the original hand-derived discrete examples. It also confirmed that, at fixed genome supply, channel obstruction partially opposes rather than compounds the companion paper’s (Davis 2026) stochastic multi-occupancy mechanism. The author reviewed all AI-assisted calculations, figures, and interpretations for consistency with the hand analysis and with the assumptions and claims stated in the text. The full derivation is provided in the Appendix.

3. Results

3.1. Channel Availability at the Canonical Ratio

At the canonical ratio f_VP3 = 50/60 ≈ 0.833, P(open) = 0.833⁵ ≈ 0.40. The mean number of open channels per capsid is 12 × 0.40 ≈ 4.8, and the probability that a capsid has at least one open channel is 1 − (1 − 0.40)¹² ≈ 0.998. Almost every capsid can package; the probability of the all-twelve-open particle is 0.40¹² ≈ 2 × 10⁻⁵.

Figure 1. Distribution of the number of open (all-VP3) channels per capsid, B(12, f_VP3⁵), at the canonical 1:1:10 ratio (blue) versus the skewed 1:1:2 ratio (Lecomte et al.). At 1:1:2, the distribution collapses toward zero open channels.

Figure 1. Distribution of the number of open (all-VP3) channels per capsid, B(12, f_VP3⁵), at the canonical 1:1:10 ratio (blue) versus the skewed 1:1:2 ratio (Lecomte et al.). At 1:1:2, the distribution collapses toward zero open channels.

3.2. Coupling to the Stochastic Ceiling

Although it might seem that the two statistical constraints should be multiplied (open-channel fraction times the single-genome probability), they are not, because the open-channel fraction (0.40) is the proportion of open channels, not the proportion of capsids that can package, and a capsid needs only one open channel. The correct capsid-level availability factor (decimal fraction of one open channel) at the canonical 1:1 ratio is therefore ~0.998.

At the canonical ratio, the combined ceiling for genome supply is ~38.4% authentic single-genome capsids and essentially identical to the all-channels-open case, because a higher genome supply (π ≈ 0.21 versus 0.08) fully compensates for fewer open channels. Channel obstruction at the canonical ratio appears therefore to have little negative effect.

The obstruction effect becomes most noticeable at a lower threshold. Because P(open) = f_VP3⁵ falls steeply as the VP3 mole fraction drops, the fraction of capsids able to package collapses once VP1/VP2 are over-incorporated. The “knee” is near f_VP3 ≈ 0.75: above it, essentially all capsids can package, and the maximum authentic fraction holds at ~38%; below it, empties dominate. At a 1:1:2 ratio (f_VP3 = 0.50), the mean open-channel count is 0.4. About 68% of capsids have no open channel; only ~32% can package, and the maximum authentic fraction falls to ~26%. This matches the ~70% empty behavior observed for the skewed-ratio case presented in the original 2020 poster.

Figure 2. Open-channel fraction (f_VP3⁵), the percentage of capsids with at least one open channel, and the maximum achievable authentic fraction, as functions of the VP3 mole fraction. The threshold near f_VP3 ≈ 0.75 separates a safe plateau from steep packaging failure; canonical (1:1:10) and skewed (1:1:2) ratios are indicated.

Figure 2. Open-channel fraction (f_VP3⁵), the percentage of capsids with at least one open channel, and the maximum achievable authentic fraction, as functions of the VP3 mole fraction. The threshold near f_VP3 ≈ 0.75 separates a safe plateau from steep packaging failure; canonical (1:1:10) and skewed (1:1:2) ratios are indicated.

The track of this coupling is qualitatively more important than its absolute magnitude. At a fixed genome supply, a population with fewer open channels produces a lower fraction of multi-occupancy (Head-Full) defectives than a fully open population, but at the cost of a higher empty-capsid fraction. This result follows directly from the reduced number of entry routes: fewer open channels reduce the probability that multiple genomes enter the same capsid. Thus, channel obstruction does not add to the stochastic multi-occupancy mechanism described in the companion paper in a simple multiplicative manner. Instead, surprisingly, the two effects partially counterbalance one another. In practical terms, channel obstruction reduces stealth-defective particles only by shifting part of the defect burden toward empty capsids.

Figure 3. At matched genome supply (per-channel loading probability π), channel obstruction at the canonical ratio lowers the multi-occupancy (Head-Full) fraction. It increases the empty fraction relative to that of a fully open capsid. The two mechanisms oppose each other rather than compound.

Figure 3. At matched genome supply (per-channel loading probability π), channel obstruction at the canonical ratio lowers the multi-occupancy (Head-Full) fraction. It increases the empty fraction relative to that of a fully open capsid. The two mechanisms oppose each other rather than compound.

These findings indicate that VP1/VP2-mediated channel obstruction acts primarily as a stoichiometry-dependent threshold rather than as a second universal ceiling on encapsidation. At wild-type-like VP ratios, the effect is predicted to be largely silent and may reduce multi-occupancy by limiting the number of available entry routes. In contrast, over-incorporation of VP1 and VP2 is predicted to make this mechanism a dominant source of empty capsids.

4. Discussion

This paper and its companion (Davis 2026) identify two distinct contributors to AAV encapsidation failure: the companion paper defines a stochastic ceiling on authentic single-genome packaging, whereas the present work proposes a structural, stoichiometry-dependent obstruction of genome-entry channels. The main advance is revealing how these mechanisms interact under dynamic packaging conditions and showing that they do not combine multiplicatively to impose a lower full-capsid ceiling. At canonical VP stoichiometry, channel obstruction is predicted to be nearly silent, and at fixed genome supply, it partially opposes stochastic multi-occupancy by reducing the number of available entry routes. The obstruction mechanism becomes consequential only beyond a stoichiometric threshold, when VP1/VP2 over-incorporation drives a large fraction of capsids toward zero open channels.

Available structural studies primarily describe mature, post-packaging AAV particles, whereas the transient packaging intermediate remains incompletely resolved. The hypothesis proposed here concerns this early encapsidation state and predicts a steep dependence of the empty-capsid fraction on the VP3 mole fraction. This prediction is experimentally testable without first resolving the precise timing of N-terminal occupancy: systematic titration of VP1/VP2 expression, followed by quantitative measurement of empty, singly packaged, and defective particles, should reveal whether empty-capsid formation increases nonlinearly as the VP3 fraction falls.

This proposal is intended to extend, rather than displace, prior published work. The stochastic-assembly model of VP composition is derived from Wörner et al. (2021); the channel-availability binomial developed here applies that model to the fivefold-pore problem. Likewise, the effect of VP stoichiometry on potency is well established, whereas the contribution proposed here concerns packaging efficiency and empty-capsid formation. VP3-only assembly, VP-reduced genome packaging, and surface rescue of infectivity have also been demonstrated (Grieger et al. 2007, Warrington et al. 2004). The present contribution integrates these insights into a quantitative threshold model and reconsiders the engineering rationale for a single-protein-capsid vector.

4.1. Evidence

VP3-only and VP-reduced particles provide the key experimental basis for the manufacturing enhancement considered below. Particles assembled without VP1, or from VP3 alone, can form authentic capsids, package genomes, and, when VP1-associated functions are supplied from the capsid surface, restore infectivity (Warrington et al. 2004). More directly, Grieger et al. (2007) showed that a surface-exposed VP1-NLS fusion rescues the infectivity of otherwise noninfectious VP2/VP3 and VP3-only capsids but does not rescue fivefold-pore mutants. These findings highlight two key points for the proposed design strategy: VP1/VP2 N-terminal extensions are not required for genome packaging itself, and their essential post-packaging functions can, in principle, be repositioned to an external capsid site rather than retained as intraluminal peptides threaded through the penton pore.

VP stoichiometry is a “quality attribute” that affects potency. Deviations from 1:1:10 alter transduction and are increasingly treated as a critical quality attribute (Maruno et al. 2025, Wörner et al. 2021). Therefore, the model’s testable predictions about packaging (empty fraction) and the threshold dependence of the empty fraction on the VP3 mole fraction have both therapeutic and manufacturing relevance.

The pervasive, serotype-independent excess of empty capsids (Penaud-Budloo et al. 2018) is conventionally attributed to kinetic uncoupling of assembly and replication. The threshold model described here offers a complementary, stoichiometry-dependent insight that is additive to the kinetic one and whose validity can be demonstrated through VP-ratio titration experiments.

Structural localization of the N-termini. Cryo-electron microscopy places VP1/VP2 N-terminal density inside the capsid and, in mature particles, within the fivefold pore (Kaelber et al. 2025, Kronenberg et al. 2005). A recent AAV5 study resolves a rod-like density in the channel but, most plausibly, assigns it to the VP3 N-terminus and reports possible pore-to-pore heterogeneity, tentatively suggesting a specialized genome pore (Gliwa et al. 2025). The unresolved questions (Section 1.5) are therefore two, namely the timing of N-terminal occupancy relative to packaging and the identity of the peptide resident in any given pore.

4.2. Implications

If the threshold model is correct, several implications follow. First, the empty-capsid burden includes a structural, stoichiometry-dependent component that is independent of genome supply and therefore cannot be eliminated by genome-side optimization alone. Second, the VP1:VP2:VP3 ratio should be treated as a critical quality attribute because its effect on empty-capsid formation is nonlinear: above f_VP3 ≈ 0.75, the system remains on a relatively safe plateau, whereas below that threshold, empty-capsid production increases steeply. Third, production conditions or vector designs that shift the capsid pool toward higher VP1/VP2 content should disproportionately increase empty-capsid production. Fourth, VP-ratio analytics, including mass spectrometry and CE-SDS, have predictive value rather than merely descriptive value. Fifth, natural differences in VP stoichiometry among serotypes may contribute to serotype-specific differences in packaging efficiency. Importantly, mild channel obstruction may not necessarily be harmful at fixed genome supply because it can reduce multi-occupancy defectives even as it increases empty capsids; in practical terms, this shifts part of the defect burden from stealth-defective particles to empty particles, which are more readily separated by common density-based purification methods.

4.3. A Reconsidered Remedy: The VP3-Only, Surface-Engineered Capsid

The hypothesis suggests a potential vector design improvement: remove VP1 and VP2 from the assembling capsid to keep all twelve fivefold channels unobstructed, while restoring their essential post-packaging functions by displaying PLA2 and NLS activities at an external capsid site, such as the VRIV loop, away from the genome-entry channel (Tseng and Agbandje-McKenna 2014). Two lines of precedent support this strategy. First, the VRIV loop tolerates large insertions: the approximately 236-residue fluorescent protein mCherry has been inserted into VRIV without loss of particle production, infectivity, or fluorescence (Judd et al. 2012). Similarly, comparably large enzymes, including β-lactamase, have been displayed on the capsid surface while preserving assembly and titer. Thus, insert size is unlikely to be the primary constraint. Second, and more directly, Grieger et al. (2007) showed that a surface-displayed VP1-NLS fusion restores infectivity to VP3-only capsids, addressing the most critical feasibility issue.

This corrected analysis also revises the VP3-only rationale proposed in the earlier foundational poster (Davis 2022). In the present model, a VP3-only capsid does not increase the authentic-yield ceiling: at the canonical ratio, the one-genome “full” ceiling is already approximately 38%, and opening all channels does not raise it. Moreover, at a fixed genome supply, a fully open capsid is predicted to produce more Head-Full multi-occupancy defectives than an obstructed capsid (Section 3.2). Therefore, a VP3-only design must be paired with the supply-control strategy to achieve particularly low instantaneous MOE [described in the companion paper (Davis 2026)]; otherwise, it would be trading empty particles for stealth-defective particles. The principal advantages of a VP3-only, surface-engineered capsid therefore lie elsewhere.

A design of this kind would be insensitive to VP-ratio drift because no VP-stoichiometry threshold would remain to be crossed. It would also yield a homogeneous single-protein capsid, thereby simplifying quality control, and could provide more uniform dosing of PLA2/NLS functions, potentially approaching 60 copies per particle rather than the approximately five copies supplied by VP1 at the canonical ratio. Most importantly, it would remove VP stoichiometry as a process-development variable. These advantages would be substantive, although they differ from the original poster’s rationale of increasing the empty-versus-full yield ceiling.

Designing the engineered VP3. Faithful reconstitution of infectivity from the loop requires more than relocating the PLA2 and an NLS, as the VP1-unique (VP1u) region shows. Beyond its contiguous PLA2 domain (the AAV PLA2 is a single, contiguous catalytic module, not a split one) and three clusters of basic residues, the VP1u carries additional conserved, infection-obligatory motifs: a YXXQ endosomal-sorting/trafficking motif (mapped to approximately residues 79–82 of VP1u), additional tyrosine-based motifs, and PDZ-binding motifs at the extreme N-terminus implicated in nuclear uptake (Popa-Wagner et al. 2012). An engineered cassette intended to restore infectivity must therefore port this set of elements into the loop, including at a minimum the endosomal-targeting tyrosine motif(s), the phospholipase, and a nuclear-localization function. Because VRIV tolerates large insertions (above), assembling such a multi-motif cassette is feasible in principle; the design question is which elements are individually necessary, not whether there is room for them.

Two specific substitutions are proposed. First, the three native basic regions of VP1u, although capable of contributing to nuclear localization, are relatively weak and context-dependent NLS elements; replacing them with a single well-characterized, high-affinity NLS, such as an SV40-type signal, could increase nuclear delivery per particle and thereby improve potency. Second, the native AAV PLA2 domain could be replaced with a well-characterized heterologous secreted PLA2 to improve biochemical robustness and provide more readily defined enzymatic activity. This substitution would not be motivated primarily by size, because the AAV VP1u PLA2 domain and small secreted PLA2 enzymes, including bee-venom-type sPLA2, are broadly comparable in size. Rather, the rationale is functional standardization. The AAV PLA2 domain is structurally related to canonical secreted phospholipases, and parvoviral PLA2 domains have been shown to be functionally interchangeable within an AAV capsid context (Girod et al. 2002, Hull et al. 2025, Popa-Wagner et al. 2012, Zadori et al. 2001).

Three caveats apply to substituting the PLA2 element. First, the extent to which PLA2 activity contributes to AAV endosomal escape remains unresolved. Recent work has emphasized AAVR-dependent trafficking through the trans-Golgi network and has questioned whether direct lytic escape from endosomes is required; one study reported that intracellular trafficking can proceed independently of PLA2 activity (Cabanes-Creus et al. 2025). Therefore, the functional contribution of any engineered phospholipase to transduction would need to be established experimentally. Second, a heterologous enzyme, particularly one derived from venom, could introduce immunogenicity or toxicity risks that would require careful evaluation before therapeutic application. Third, the selected sPLA2 would need appropriate calcium dependence, pH optimum, stability, and structural accessibility to support the required biological activity within the targeted intracellular compartment.

The remaining concerns are straightforward: whether the VP3-VRIV cassette subunit assembles efficiently; which of the ported VP1u trafficking motifs are individually necessary; whether the displayed phospholipase retains activity in the relevant compartment; whether the displayed NLS is import-competent; and whether the immunological and therapeutic profiles differ meaningfully from those of the wild type.

5. Conclusions

VP1 and VP2 N-terminal extensions may obstruct fivefold genome-entry channels, leaving only all-VP3 pentons competent for packaging and causing the number of open channels per capsid to follow B(12, f_VP3⁵). This mechanism does not impose a second multiplicative ceiling on genome encapsidation. At the canonical 1:1:10 VP ratio, nearly all capsids are predicted to retain at least one open channel. Consequently, the maximum authentic single-genome yield remains close to that predicted in the companion paper (~38%), and, at fixed genome supply, channel obstruction is predicted to reduce rather than compound Head-Full multi-occupancy defects. The major adverse effect is threshold-dependent: below f_VP3 ≈ 0.75, empty capsids become predominant, and at a 1:1:2 VP ratio, only ~32% of capsids are predicted to be packaging competent. These results identify the VP1:VP2:VP3 ratio as a critical quality attribute with nonlinear threshold behavior. They also refine the rationale for a VP3-only, surface-engineered capsid. Such a particle would display a reconstituted VP infectivity tract in an insertion-tolerant surface loop, such as VRIV, incorporating at minimum an endosomal-targeting motif, a contiguous phospholipase domain, and a strong nuclear-localization signal. This design is expected to improve particle homogeneity, robustness to VP-ratio drift, and uniform dosing of infectivity-related functions, but it is not expected to increase the intrinsic ceiling for single-genome capsid production. Finally, the proposed channel-occlusion mechanism is experimentally testable through systematic VP-ratio titration.