Recurrent and novel evolutionary pathways drive in vitro HIV-1 lenacapavir resistance with diverse phenotypic consequences

Domain: chemistry
Published: 2026-03-07
Editorial outcome: minor_revision

Abstract
The small-molecule inhibitor lenacapavir (LEN) has firmly established the HIV-1 capsid as a
promising clinical target. However, early studies have shown that HIV-1 can develop resistance
to LEN by acquiring capsid mutations. These mutations are restricted to the drug-binding pocket,
but the breadth of the virus’s capacity to evolve under LEN-directed pressure remains
incompletely understood. Here, we employ an accelerated evolution approach to probe HIV-1’s
capacity for LEN resistance. Despite strong sequence conservation across thousands of clinical
strains, two positions within the binding pocket exhibit high mutational tolerance and diverse
resistance profiles. These low-level resistance mutations recurrently select the high-level
resistance mutation M66I and together with further mutations, synergistically enhance viral fitness
by restoring nuclear entry. Subsequently, multiple charge-reversing substitutions distal to the
binding pocket emerge. Located at inter-hexamer interfaces, these substitutions modulate capsid
stability and mitigate LEN’s capsid-disrupting effects. A highly resistant variant carrying the E213K
substitution becomes drug dependent; it requires LEN to generate infectious particles. As the
novel pathways observed here may represent natural extensions of recurrent ones, this work
supports surveillance and forecasting efforts while guiding further drug development to sustain
the capsid as a viable antiviral target.

Introduction
Lenacapavir (LEN) is a small-molecule compound developed by Gilead that targets the viral
capsid, blocking HIV-1 infection by interfering with both early and late stages of the viral replication
cycle1. Its mechanism of action involves direct binding to the interface of two monomers of the CA
hexamer of HIV-1, the basic building block of the capsid lattice1,2. Clinical testing of LEN yielded
encouraging safety and efficacy outcomes, and led to its approval as the first-in-class capsid
inhibitor for highly treatment-experienced individuals living with multidrug-resistant HIV-1
infection3. LEN, a long-acting agent, has been shown to be potent in preventing HIV-1 infection4,5,
thus garnering attention as a transformative tool in global HIV prevention efforts.

Despite its picomolar potency, LEN—like all other antiretroviral agents—faces the challenge of
viral resistance. The relative sequence conservation of CA, attributed to its stringent functional
and structural requirements6,7, is often cited as the basis for LEN's high genetic barrier to
resistance. Indeed, amino acid residues near the LEN binding site in CA are highly conserved,
with sequence identity ranging from 94% to 100% across major HIV-1 subtypes6-8. However, HIV-
1 does develop LEN resistance in both in vitro and in vivo settings1,9-13. Most amino acid
substitutions associated with drug resistance mutations (DRMs) are located near the LEN binding
site and confer resistance levels ranging from a few-fold to over 1,000-fold. High-level DRMs such
as the well-known M66I substitution often come at a significant cost to viral replicative fitness;
however, they still emerge in individuals treated with LEN12. Although clinical trials have
highlighted LEN’s strong potential as a pre-exposure prophylaxis (PrEP) agent4,5, two individuals
who acquired HIV-1 during LEN-based PrEP carried the LEN-resistant N74D variant5. Given that
N74D is rarely observed among LEN-untreated HIV-1-infected individuals14, this is most
consistent with de novo evolution rather than selective transmission.

While both in vitro and clinical studies have revealed LEN-resistant mutations, the extent of HIV-
1’s ability to acquire higher-level resistance remains incompletely understood. It is plausible that
the virus has not yet fully exploited this potential due to several factors. First, extensive studies
on HIV-1 CA biology, including viral escape from antiviral pressures, have demonstrated the
virus’s capacity to adapt to hostile conditions through mechanisms such as altering binding sites
or modulating capsid stability15. These findings underscore that while CA is generally genetically
fragile16, it also possesses phenotypic flexibility that the virus can leverage to overcome antiviral
interventions. Second, many DRMs identified during early stages of in vitro or in vivo evolution
are single amino acid substitutions. However, recent studies, including findings from the
CAPELLA trial, have revealed that LEN resistance can evolve into more complex mutational
patterns involving multiple substitutions12. This mirrors the resistance trajectories observed with
other drug classes, such as diarylpyrimidine analog non-nucleoside reverse transcriptase
inhibitors and integrase strand-transfer inhibitors, where resistance accumulates through
combinations of mutations, particularly in the context of pre-existing variants17,18. Currently, LEN
is approved for use in a specific subset of people living with HIV-1, which has limited its
widespread application. However, as its use expands in both treatment and prevention settings,
the selective pressure on the virus will increase—potentially accelerating the emergence and
evolution of resistance.

In this study, we aimed to uncover how HIV-1 circumvents LEN pressure at the earliest mutational
steps and acquires high-level resistance. A multivariant selection and adaptation strategy allowed
us to examine evolutionary trajectories and the underlying drivers of resistance. Our results
identified known and previously unrecognized resistance pathways, offering insights into the
adaptive potential of the HIV-1 capsid as well as diverse mechanisms and phenotypic
consequences of LEN resistance.

Results
Mutational tolerance in conserved positions of HIV-1 CA
Among several DRMs reported so far, we focused on positions 67 and 74 in this study, as single-
nucleotide mutations at these positions—Q67H and N74D—frequently emerge in individuals
receiving LEN1,11,12. We used site-saturation mutagenesis to probe less-accessible multi-
nucleotide DRMs which often emerge in clinical settings under selective pressure from other
antiretrovirals, rather than those selected in vitro17,19. As controls, we included two additional
positions (4 and 18) that influence capsid stability20,21 (Fig. 1). These amino acid residues are
highly conserved across thousands of HIV-1 strains (Fig. 1b, right panel).

The multiplex variant selection assay consists of two stages: the first phase selects for infection
conditions where ~1% of target cells are infected with HIV-1 as determined by virus-encoded GFP
reporter, followed by the second viral outgrowth phase in the absence of LEN. (Fig. 1a). Sequence
analysis of variants selected five days post-expansion revealed highly variable degrees of
mutational tolerance in these four positions, with an unexpected diversity of viable variants found
in three of the four mutant libraries using an LAI backbone (Fig. 1b, left panel and Supplementary
Fig. 1). The frequency of each individual variant ranges from WT-level to stop codon levels, which
would yield non-infectious virus and serve as assay background (Supplementary Fig. 1). Position
74 showed the highest mutational tolerance, while position 18 was highly intolerant, with only one
viable variant (the WT arginine). Positions 4 and 67 exhibited an intermediate phenotype, with
approximately one-half to one-third of the variants detected at frequencies above 1%. Variant
frequencies in both Q67X and N74X mutant libraries across two independent experiments
(Supplementary Fig. 2) were highly concordant, supporting the robustness of the assay. Although
the occurrence of variants revealed by our experiments differs substantially from those in vivo,
the second most abundant variant identified in vitro was also the second most abundant in vivo
for all positions examined except for position 4 (Supplementary Fig. 3). For instance, the second
most prevalent variant in the R18 mutant library encodes a lysine residue—the same substitution
that ranks second in sequence databases—potentially reflecting the importance of a positive
charge at this position for inositol hexakisphosphate (IP6) binding and dNTP influx21-23. Another
key observation relevant to LEN (discussed further below) is the high frequency of Q67H in vitro,
which, although much less common overall, is the second most abundant variant observed in vivo
(Supplementary Fig. 3). These observations underscore the unexpectedly high mutational
tolerance of the LEN-binding region, which contrasts with its strong sequence conservation in
natural isolates.

Contrasting LEN resistance potentials of positions 67 and 74
The unexpectedly high mutational tolerance at positions 67 and 74—key residues involved in LEN
binding24 —has important implications for the evolution of LEN resistance. To address this, we
examined how LEN affects the enrichment of each variant in the Q67 and N74 mutant libraries
(Supplementary Fig. 4a). Both exhibited shifted dose-response curves, consistent with reduced
sensitivity to LEN. While the Q67 library displayed greater overall resistance (Supplementary Fig.
4b), the N74 library exhibited higher infectivity in a drug range which suppresses WT infection by
more than 10-fold. (Supplementary Fig. 4b). Sequence analysis of variant abundance following
drug selection revealed strikingly distinct mutational profiles between positions 67 and 74 (Fig. 2).
In the N74 mutant library, aspartic acid (N74D) became dominant after selection with 3.1 nM LEN.
This well-characterized mutation confers LEN resistance in vitro and has been observed in
individuals treated with LEN11,12. A similar charge-altering substitution, N74E (glutamic acid), also
increased in frequency and conferred resistance when tested in a cloned virus (Figs. 2a and S4c).
A 2D plot comparing amino acid frequencies in the presence and absence of LEN demonstrated
a clear distinction, with N74D and N74E deviating from the other variants (Fig. 2a). The Q67
mutant library exhibited a contrasting resistance pattern (Fig. 2b). This library contained fewer
viable variants (~7) than the N74 mutant library. However, most of these viable variants in the
Q67 library increased in abundance under the LEN-treated condition, surpassing the WT
glutamine (Q67) (Fig. 2b). LEN resistance of these variants was validated using molecularly
cloned viruses (Supplementary Fig. 4d).

Q67 variants enriched following LEN treatment included those carrying the well-known LEN-
resistant Q67H mutation. However, Q67H was outpaced by Q67W, Q67F, Q67Y, and, most of all,
Q67N (Fig. 2b). Indeed, this ranking was reflected in dose-response analyses with cloned viruses,
confirming that these variants (Q67F/N/W/Y) exhibited higher IC values than Q67H.
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(Supplementary Fig. 4d). Among these variants, a Q67N substitution was observed in one
participant from the CAPELLA study, whereas the Q67H change occurred in multiple participants
within the same study12. The higher occurrence of Q67H compared to Q67N in clinical settings
may be attributed to the high replicative capacity of Q67H in LEN-free conditions, differences in
selective pressure in vivo, and codon-based accessibility: Q67H arises from a single nucleotide
change from WT, whereas Q67N requires two. We next assessed the viral infectivity and
replicative capacity of Q67H and Q67N in primary CD4⁺ T cells. Single-round infectivity was
comparable between Q67H and Q67N, while Q67H showed a modest trend toward higher
replicative capacity relative to Q67N that did not reach statistical significance. As controls, M66I
and N74D exhibited markedly impaired infectivity and replication in primary CD4⁺ T cells
(Supplementary Fig. 5). Overall, these results reveal distinct resistance potentials of key sites and
highlight fitness and resistance profiles of several previously undescribed, less-accessible multi-
nucleotide substitutions.

HIV-1 can develop micromolar-level resistance to LEN
We next used in vitro viral adaptation to investigate the ability of HIV-1 to develop resistance to
LEN. Unlike in vivo studies, in vitro systems offer the advantage of allowing viral evolution under
controlled conditions with frequent sampling, providing detailed insights into evolutionary
trajectories. While a previous study investigated LEN resistance in vitro, it used drug
concentrations below the upper limit of physiological levels (~100 nM)1. To accelerate the
adaptation process up to and beyond this point, we started with Q67N, the most abundant variant
in our selection experiment (Fig. 2b). We monitored the proportion of virus-infected cells using a
replication-competent GFP-reporter virus. Adaptation was performed using a conventional dose-
escalation scheme, where our initial dose titration determined that a LEN concentration of 10.9
nM maintained a low but substantial level of GFP-positive cells (~5%) for the Q67N variant (Fig.
3a). After approximately 10 days of culture, GFP-positive cells rose above 10%. In response, we
increased the LEN concentration to 21.9 nM, which was soon followed by a more pronounced
breakthrough (>50% GFP-positive cells). The supernatant from this culture was used to initiate a
second round of adaptation, now requiring 43.8 nM LEN to maintain a steady level of virus
infection. Adhering to this scheme, the culture progressed through 175, 350, 700, and finally 2800
nM of LEN in about forty days. Remarkably, the virus exhibited breakthrough even at micromolar
LEN concentrations—over 10,000-fold higher than the IC of LEN against the WT virus.
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To quantify changes in LEN sensitivity of the adapted viruses, we generated dose-response
curves using viral supernatants harvested at different time points (Fig. 3b). The starting Q67N
virus exhibited ~17-fold resistance relative to WT. After 18 days of adaptation, the virus showed
further resistance, with a 37-fold increase in IC . Virus harvested at 175 nM LEN showed a
50
dramatic increase, with an IC of ~490 nM (~1500-fold over WT). The most resistant variant
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exhibited an IC >1 µM. These results demonstrate that HIV-1, given the right combination of
50
mutation and selective pressure, can evolve resistance even at micromolar concentrations of LEN.

Emergence of M66I and CTD mutations at CA hexamer interfaces during HIV-1 adaptation
to LEN
Analysis of CA sequences from adapted viruses revealed a stepwise accumulation of both known
and novel amino acid substitutions, which increased in frequency alongside rising drug
concentrations (Fig. 4a). Throughout the adaptation process, the original Q67N mutation
remained consistently present. A key substitution that emerged at 21.9 nM LEN was M66I—a
well-characterized resistance mutation previously linked to high-level LEN resistance in both in
vitro studies and in HIV-1 variants from LEN-treated individuals1,12. The frequency of this
substitution increased from 55% to 64% between 11 and 14 days post selection. An independent
adaptation experiment similarly selected for M66I, reaching 71.4% at 21.9 nM (Supplementary
Fig. 6). These parallel outcomes highlight M66I as a recurrent solution to LEN pressure by viruses
carrying the Q67N substitution.

By 175 nM LEN, the adapted population showed three major differences from the earlier 21.9 nM
population (Fig. 4). First, the frequency of M66I decreased to 63.2%, coinciding with the
emergence of another high-level resistance mutation, L56I (36.6%). The linkage analysis revealed
that these two changes were mutually exclusive, indicating that nearly all Q67N-containing
variants (99.5%) carried either L56I or M66I, but not both (Supplementary Fig. 7). Second, T107N,
a well-documented DRM1, appeared at moderate frequency (7.0%). Third, a novel substitution,
E187K, which has not been described as a DRM for LEN, emerged at high frequency (42.1%).
Interestingly, E187K appeared alongside several other CTD mutations, most involving
substitutions to lysine: N183K, T210K, and E213K. While the frequency of E187K remained
relatively stable across increasing LEN concentrations (42.1% at 175 nM, 36.9% at 700 nM, and
34.1% at 2800 nM), the other CTD substitutions—particularly E213K—continued to increase,
reaching 46.7% at 2800 nM. Importantly, these CTD substitutions, along with E75K (another
charge-altering substitution), are located at the interfaces between CA hexamers; N183K and
E187K map to the dimeric interface, while E75K, T210K, and E213K reside at the trimeric
interface (Fig. 4b and Supplementary Fig. 8). Thus, viral adaptation selected for amino acid
substitutions both at the drug-binding pocket and at the inter-hexamer interfaces of the CA CTD,
which likely cooperate to enable high-level LEN resistance.

Q67N facilitates M66I emergence by providing low-level resistance and fitness
compensation. We first assessed the effects of M66I, which emerged early during adaptation at
low LEN concentrations. Experiments using infectious molecular clones revealed that the
acquisition of M66I by Q67N increased the IC of LEN by more than 500-fold (Fig. 5a). M66I has
50
been shown to severely compromise viral infectivity1,10, as confirmed by our particle-normalized
infectivity assay (Fig. 5b), and it is typically accompanied by additional DRMs in clinical samples12.
This suggests that the infectivity defect of M66I likely limits its adaptation unless compensatory
mutations are present. Indeed, Q67N substantially restored the infectivity of M66I (Fig. 5b).
Furthermore, other low-level DRMs, such as Q67H and A105T, also improved the infectivity of
M66I when combined (Fig. 5b). Strikingly, low-level DRMs in combination with M66I are more
susceptible to LEN than M66I on its own (Fig. 5c). This was particularly evident for A105T (Fig.
5c). These results suggest that the role of Q67N shifts from a primary resistance determinant to
facilitating the emergence and stabilization of the high-level resistance mutation M66I. Thus,
Q67N and other low-level DRMs likely serve as enablers of stronger resistance pathways in the
evolution of drug-resistant HIV-1.

E187K mutation augments high-dose LEN resistance
After acquiring M66I, the virus gained further resistance while approaching the maximum
physiological concentration of LEN (100–200 nM)5,25. The key amino acid substitution that
significantly increased in frequency was E187K (Fig. 4), a change not previously reported in LEN
resistance literature. E187K emerged at similar frequencies in combination with either M66I or
L56I. An infectious molecular clone of M66I+Q67N+E187K confirmed increased resistance to
high-dose LEN (Fig. 6a). The effect of E187K on resistance appears to be context-dependent as
E187K alone displays WT LEN sensitivity, if not further susceptibility (Fig. 6b). E187K did not
modulate increased resistance when combined with another individual key DRM at a time
(Supplementary Fig. 9a). Both double and triple mutants impair the infectivity (Fig. 6c), also
supporting the notion that these changes arose to increase the resistance, but not viral fitness.
Interestingly, N183K, which emerged after high-dose selection and resides close to E187K, also
did not affect LEN sensitivity (Supplementary Fig. 9b). Thus, the resistance-enhancing effect of
E187K is unlikely to result from altered drug binding.

E187K enhances core stability and counters core collapse at high LEN doses
E187K is adjacent to residues M184 and W185, which constitute part of the dimeric interface
between CA hexamers. Thus, E187K may influence capsid stability and disassembly, contributing
to enhanced LEN resistance. To investigate this, we assessed capsid stability and integrity using
cell-free assays. Capsid stability was assessed by measuring core yield after isolation using a
spin-through method (Fig. 7a). The results showed that E187K exhibited a ~3-fold increase in
core yield compared to WT virus (Fig. 7b and 7c). We also used a previously established in vitro
core disassembly assay with minor modifications26,27. Isolated cores were incubated for 1 hour at
4 or 37°C, then spun down to measure the levels of pelletable cores, which represent cores with
intact integrity. Incubation at 37°C drastically reduced the pelletable cores (Fig. 7d and 7e).
However, incubation with 100 µM of inositol hexakisphosphate (IP6) counteracted this reduction
and restored the level of pelletable cores (Fig. 7e). Importantly, the E187K variant exhibited a
significantly smaller reduction in pelletable cores upon incubation at 37°C without IP6 compared
to the WT virus (Fig. 7e).

Additionally, we compared the triple mutant (M66I+Q67N+E187K) with the precursor double
mutant (M66I+Q67N). Interestingly, the double mutant (M66I+Q67N) already showed higher core
yields compared to the WT virus, and there was no significant difference in core yield between
the double and triple mutants (Fig. 7c). Similarly, core disassembly phenotypes observed by both
mutants showed a lower magnitude of disassembly at 37°C compared to WT, with no significant
difference between the double and triple mutants (Fig. 7e).

We then directly tested the effects of LEN on core integrity by incubating isolated cores in
increasing concentrations of LEN (Fig. 7f). As expected from known core-disrupting activity by
LEN28,29, LEN treatment resulted in a steady decrease in pelletable cores of the WT virus up to
20 nM, at which the curve plateaued (Fig. 7g). Consistent with their LEN resistance phenotypes,
both the double and triple mutants preserved pelletable cores at low to mid-nanomolar
concentrations (4, 20, and 100 nM; Fig. 7g). Remarkably, the triple mutant differed from the double
mutant at the highest concentration; at 500 nM, the level of pelletable cores was significantly
higher for M66I+Q67N with E187K than without it (Fig. 7g). Taken together, these results indicate
that E187K, which stabilizes viral cores, counteracts the core-collapsing activity induced by high
LEN doses in a context-dependent manner.

Convergent evolution drives shared selection of M66I and CTD lysine increase
We have shown so far that adaptation of the Q67N mutant to elevated LEN doses selected for
well-described high-level DRMs, including M66I and L56I, as well as previously unreported
mutations in the CTD of CA (Fig. 4). To investigate how the initial DRMs influence evolutionary
trajectories in drug resistance, we performed viral adaptation using a pooled set of LEN-resistant
Q67 variants (excluding Q67N)—specifically, Q67H, Q67F, Q67W, and Q67Y (Fig. 8).

A similar dose-escalation scheme (Fig. 8a) to Q67N was employed for viral adaptation of the
pooled variants (Q67F/H/W/Y) and successfully selected for variants with increased LEN
resistance (Fig. 8b). NGS analysis of viral sequences revealed both shared and distinct
evolutionary patterns in the pooled variants relative to Q67N evolution (Fig. 8c, Supplementary
Fig. 10). Selection at the initial 12.5 nM time point (shown as 12.5a in Fig. 8c) primarily enriched
the Q67W variant (62.9%), followed by Q67H (22.9%) and Q67Y (14.2%), with Q67F completely
eliminated. However, from this point forward, all emerging variants retained the M66I+Q67H
combination and accumulated additional mutations (Supplementary Fig. 10). Both substitutions
reside within the drug-binding site and were maintained at higher drug concentrations. A notable
de novo mutation that emerged during selection of the M66I+Q67H background was the well-
known LEN resistance mutation N74D, which became nearly fixed at 200 nM; continued selection
at 400 nM subsequently led to acquisition of A77V, resulting in the M66I+Q67H+N74D+A77V
genotype. Importantly, the combination of these three mutations (M66I, Q67H, and N74D),
together with another low-level DRM (A105T), exactly matches the mutations observed in one of
the LEN-treated individuals from the CAPELLA study12.

Acquisition of high-level resistance to LEN via these changes was confirmed using cloned viruses
(Fig. 8d). The two adapted variants found here (M66I+Q67H+N74D, with or without A77V) 
exhibited an increased level of resistance (Fig. 8d). The levels of resistance match with those of
variants identified in two specific participants in the CAPELLA study (M66I+N74D+A105T, with or
without Q67H; Supplementary Fig. 11a). This magnitude of the increase in LEN resistance by
these Q67H variants is well above the resistance level of Q67H alone, which has a 4.7-fold
increase in IC . Similar to the viral adaptation of Q67N, the combinations of these DRMs restored
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viral infectivity, particularly when compared to the M66I mutation (Supplementary Fig. 11b). This
supports the idea that compensatory mutations were selected to enhance viral replicative capacity
rather than to further increase drug resistance.

Further selection at 400 and 800 nM revealed both shared and distinct evolutionary trajectories
between Q67N and Q67H (Figs. 4 and 8). Specifically, the CTD lysine substitutions E187K and
E213K—previously observed during Q67N adaptation—emerged and increased in frequency with
escalating drug doses (Fig. 8c). Different from the Q67N adaptation, M66I+Q67H acquired
additional binding-site mutations before (N74D) or alongside (A77V) CTD substitutions; the
variant M66I+Q67H+N74D+A77V comprised 76.2% of reads before E187K was even detected.
These results reveal both convergent and divergent evolution during the viral adaptation of Q67
mutants to high-LEN concentrations, sharing the initial selection of M66I and later novel CTD
substitutions.

Combinatorial mutations drive restoration of HIV-1 infectivity in non-dividing cells during
in vivo LEN resistance evolution
One key property impaired by the high-level DRM M66I is HIV’s ability to efficiently infect non-
dividing cells, as shown by Briganti et al.30 and confirmed by our results (Fig. 9). The severe
infectivity defect in non-dividing cells was shared by another high-level LEN resistance mutation,
L56I, which likewise shows strong impairment in non-dividing cells (Supplementary Fig. 12a). As
described above, we found that low-level DRMs can compensate for the fitness cost imposed by
M66I. We therefore asked whether any of these mutation combinations could also restore the
ability of M66I-containing variants to infect non-dividing cells. Q67N retains the ability to efficiently
infect non-dividing cells; however, neither it nor the E187K mutation rescued the M66I mutation’s
infectivity in non-dividing cells (Fig. 9a and 9b). Other low-level DRMs, such as Q67H and A105T,
also failed to do so (Supplementary Fig. 12b).

In marked contrast, M66I infectivity in non-dividing cells was rescued to varying degrees by triple
(M66I+Q67H+N74D) and quadruple (M66I+Q67H+N74D+A77V) mutants that emerged in our
Q67F/H/W/Y adaptation as well as triple (M66I+N74D+A105T) and quadruple
(M66I+Q67H+N74D+A105T) mutants seen in two specific CAPELLA study participants12 (Fig. 9c
and 9d). The recovery of infectivity in non-dividing cells was more pronounced in the two clinically
observed mutants. In particular, the quadruple mutant (M66I+Q67H+N74D+A105T)12 almost
completely restored infectivity in non-dividing cells (Fig. 9c and 9d). Together, these results
highlight the divergent evolution driven by the initial mutation, which has a profound effect on the
phenotypic outcomes of adapted variants, and underscore the importance of multiple amino acid
replacements in fully restoring the ability to infect non-dividing cells—an effect critically observed
in an in vivo viral evolution setting.

LEN-dependent infectivity conferred by E213K
In addition to E187K, another glutamic acid–to–lysine (E-to-K) substitution in the CTD—E213K—
also commonly emerged at high LEN doses in two adaptation experiments (Figs. 4 and 8). Various
E213K-containing molecular clones were constructed but found to be non-infectious. As these
E213K variants emerged upon replication in high LEN concentrations, we hypothesized that
variants carrying E213K may depend on LEN for infection. Indeed, this was the case. The triple
mutant virus M66I+Q67H+E213K generated in transfections with increasing concentrations of
LEN showed increasing infectivity, contrasting the dose-dependent reduction of the WT (Fig. 10a).
Namely, the triple mutant displayed little to no infectivity from 10 pM to 1 nM LEN, followed by a
sharp, dose-dependent increase in luciferase activity peaking at 1 µM LEN. This LEN dependency
appeared to be largely specific to this mutation combination, except for the M66I+E213K double
mutant which exhibited moderate infectivity at 100–1000 nM LEN (Supplementary Fig. 13a).
This initial experiment allowed residual LEN from virus-producing cells to carry over into target
cells. To determine the stage at which the triple mutant is rescued by LEN, LEN was removed by
concentrating virus stocks and resuspending them in LEN-free medium. M66I+Q67H+E213K
viruses produced with LEN but used to infect cells without LEN showed 20–30-fold higher
infectivity than viruses made and used without LEN (Fig. 10b). In addition, LEN added only to
target cells enhanced infectivity: virus made without LEN showed an 8-fold increase, and virus
made with LEN showed a 3.8-fold increase. These results suggest that LEN in producer cells
plays a more dominant role in enabling infectivity of the triple mutant.

To further examine the role of LEN during late stages of replication, we measured virion-
associated RT activity. As expected, 1 µM LEN reduced RT activity in WT virus particles (Fig.
10c). The triple mutant showed an overall reduction in particle production, but, notably, LEN did
not reduce nor increase RT activity in this context—levels were nearly equivalent between treated
and untreated samples. These results suggest that LEN-mediated enhancement of infectivity is
not due to an increased particle production. When we examined core yields of viruses produced
in the presence or absence of the drug, LEN present during virus production drastically reduced
core yield in WT virus, likely due to capsid collapse from CA hyper-stabilization (Fig. 10d). In
contrast, the triple mutant displayed two notable features. First, in the absence of LEN, it produced
fewer cores than WT. Second, LEN addition rescued core yield to levels comparable to WT virus
produced without LEN (Fig. 10d). These findings suggest that LEN packaged into virus particles
plays a critical role in proper capsid maturation and/or stability for the triple mutant carrying E213K.
Altogether, our results demonstrate that LEN resistance at high doses can lead to strict LEN
dependence for the production of infectious virus particles.

Capsid backbone influences lenacapavir resistance evolution
Having established that several amino acid substitutions identified using the LAI-based clone are
also observed among resistance-associated changes in the CAPELLA study12, we next examined
whether the CA backbone influences the evolutionary trajectory of LEN resistance. To address
this, we generated a chimeric infectious molecular clone in which most of the Gag-coding region
was replaced with that of CH505, a subtype C transmitted/founder (T/F) strain. Using a dose-
escalation adaptation strategy analogous to that employed for LAI, we initiated selection with a
CH505 chimeric virus carrying Q67H, as this mutation was both highly prevalent and became
dominant during prior adaptation.

Sequence analysis of Q67H-containing viruses revealed acquisition of a distinct set of secondary
mutations (Figure S14). Notably, selection favored K70R, resulting in the Q67H+K70R
combination, which mirrors another mutation pattern observed in LEN-treated participants12. Upon
further escalation of drug pressure, and in contrast to our initial expectations, an additional novel
substitution (A174T) emerged in the CTD of CA. Although limited in scope, these findings suggest
that the CA backbone can shape LEN resistance pathways, while supporting our model that HIV-
1 capsid resistance to lenacapavir can arise through both recurrent and novel evolutionary routes.

Methods
All research was conducted in accordance with relevant ethical regulations. This study did not
involve human subjects research or clinical investigation. Peripheral blood mononuclear cells
(PBMCs) used in this study were obtained from leukocytes collected from anonymous donors
through the New York Blood Center. These materials were fully de-identified, and informed
consent was not applicable for their use in this study.

Chemicals. Lenacapavir (HY-111964) was obtained from MedChemExpress. Aphidicolin
(5047440001) was purchased from Sigma. Plasmocin® Prophylactic (ant-mpp) was acquired
from InvivoGen.

HIV-1 CA mutant library construction. We used pBru3ori-GFP3, a replication-competent
molecular clone of the LAI strain32. This reporter virus expresses GFP in place of the nef gene,
while all other viral genes remain intact. To facilitate mutant library construction, we generated an
intermediate plasmid in which a large portion of the gag-coding sequence was deleted. This
design minimizes contamination from undigested vector, which could produce WT virus and
confound our analysis of mutant variants that are expected to be less fit than the WT virus.

For PCR-mediated mutagenesis, we designed pairs of 27-bp mutagenized oligonucleotides
(sense and antisense) with the mutated codon (three nucleotides) positioned centrally. Each
codon position was completely randomized (NNN). Oligonucleotide primers used for site-directed
mutagenesis and other PCR-based applications were synthesized by Eurofins Genomics or
Integrated DNA Technologies (IDT). Oligonucleotide sequences used in this study are listed in
Supplementary Table 1.

PCR-mediated mutagenesis was performed using NarI or BssHII at the 5’ end and ApaI at the 3’
end. Oligonucleotide primers annealing 15–25 bp upstream or downstream of these sites were
used. Separate PCR reactions amplified the 5’ and 3’ fragments using mutagenized
oligonucleotides. Purified PCR products were mixed in a Gibson assembly reaction with vectors
digested by NarI or BssHII and ApaI. In some cases, a second PCR step was performed to
combine the two PCR fragments before cloning.

The assembled products were used for chemical transformation. We observed >300 colonies,
whereas the control transformation with digested vector alone yielded ~60 colonies. Each mutant
library, where a single amino acid residue was randomized, had a theoretical size of 64. To
achieve 99% probability of full coverage, 338 transformants were required61. Plasmid DNA for
each mutant library was prepared by scraping colonies from bacterial plates. Each plasmid DNA
library was then used for transfection.

Generation of mutant clones. Individual proviral clones carrying single or multiple mutations
were generated by either PCR amplification of Gag-encoding sequences from viral DNA of
selected or adapted variants, or by PCR-mediated mutagenesis. PCR amplicons containing
mutations were cloned into replication-competent or Env-defective infectious clones of GFP
reporter viruses. A chimeric LAI–CH505 construct was generated by replacing the Gag-coding
region between BssHII and ApaI with the corresponding sequence from the CH505
transmitted/founder virus (ARP-13550, BEI)62 using Gibson assembly. Site-directed mutagenesis
was then used to introduce Q67H into the CH505 Gag backbone. Introduction of mutations was
confirmed by Sanger sequencing (GENEWIZ) or by whole plasmid sequencing (GENEWIZ or
Plasmidsaurus). Newly generated plasmids in this study are available from the corresponding
author upon reasonable request.

Cell culture. Adherent cells, such as Lenti-X 293T cells (632180, Takara) and TZM-bl (HRP-8129,
BEI)63, were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Corning) supplemented
with 10% fetal bovine serum (FBS; Sigma) and 1× penicillin-streptomycin (P/S; Corning). MT-4
cells (ARP-120, BEI) were cultured in RPMI 1640 medium (Corning) supplemented with 10% FBS,
1× P/S, and 2 mM L-glutamine (Corning). In certain experiments, Plasmocin® prophylactic (ant-
mpp) was added to the culture medium. Primary CD4⁺ T cells were isolated from PBMCs derived
from leukocytes collected from anonymous donors through the New York Blood Center, using the
EasySep™ Human CD4⁺ T Cell Enrichment Kit (STEMCELL Technologies) and activated using
Dynabeads™ Human T-Activator CD3/CD28 (Thermo Fisher Scientific).

Viruses. Stocks of replication-competent GFP reporter viruses were generated by transient
transfection of HEK293T cells using polyethylenimine (PEI; Polysciences). For mutant libraries,
plasmid DNA libraries were co-transfected with the Gag-Pol expression vector pCRV1-Gag-Pol64
to supply WT Gag-Pol during the initial round of virus production, minimizing potential dominant-
negative effects of CA mutations. In some cases, a VSV-G expression vector was also included
to enhance the first round of infection. For the generation of pseudoviruses from Env-defective
infectious clones, a VSV-G expression vector65 was included.

High-titer virus stocks were generated by virus concentration. Briefly, 293T or Lenti-X 293T cells
were seeded in 100-mm plates at 5 × 10⁶ cells per plate. Culture medium was either left
unchanged or replaced with fresh medium one day after transfection. Supernatants were
harvested two days post-transfection, filtered using a Steriflip filter unit (Millipore), and gently
layered onto 5 mL of 20% (w/v) sucrose solution in polyallomer tubes (Beckman Coulter).
Ultracentrifugation was performed at 100,000 × g for 2 hours at 4°C using an SW28 rotor. Virus
pellets were incubated with 3.2 mL of culture medium for at least 1 hour at 4°C before
resuspension. Virus aliquots were prepared and stored at −80°C until use.

Multiplex variant selection. MT-4 cells were plated at 3 × 10⁶ cells per well in 12- or 24-well
plates and infected with varying amounts of virus. In some experiments, spinoculation was used
to enhance infection. GFP-positive cells were assessed 2- or 3-days post-infection by flow
cytometry. Virus-infected cells were then diluted with uninfected target cells to achieve 1–3%
GFP-positive cells, in the presence or absence of LEN at various concentrations. The virus was
allowed to propagate for 3 days, and viral growth was quantified by flow cytometry. Infected cells
were either harvested immediately or expanded for several additional days before harvesting for
sequencing analysis.

Viral adaptation to lenacapavir. Viral adaptation was performed using a dose-escalation
scheme. Briefly, MT-4 cells were infected with replication-competent viruses carrying different
mutations at position 67 of CA. Two main viral adaptation experiments were conducted: one using
a single Q67N mutant virus, and the other using a pooled mixture of four mutants (Q67F/H/W/Y).
For the pooled adaptation, each mutant virus was initially infected separately, and similar levels
of each variant were mixed 2–3 days post-infection. Viral adaptation was carried out to maintain
approximately 5% virus-infected cells in the presence of LEN. The initial LEN concentration was
10.9 nM for Q67N adaptation and 12.5 nM for the pooled Q67F/H/W/Y adaptation. The proportion
of virus-infected cells was monitored every 2–3 days using flow cytometry. Once an increase in
GFP-positive cells was observed, infected cells were diluted with uninfected cells to maintain ~5%
infection and exposed to increasing concentrations of LEN.

For the Q67N adaptation, a breakthrough was observed after 15 days when the proportion of
infected cells exceeded 50%. Culture supernatant was harvested, and new infections were
initiated. This process was repeated until the LEN concentration reached 2.8 µM, at which point
signs of cytotoxicity were noted. A similar dose-escalation approach was applied to the pooled
Q67F/H/W/Y viruses. At each cell split, the supernatant and a portion of the cells were collected
and stored at −80 °C.

NGS-based variant analysis. For the multi-variant selection assay, PCR primers targeting
regions containing mutagenized positions (positions 4, 18, 67, and 74) were used for amplification.
A second PCR was performed to incorporate sample barcodes and Illumina sequencing adapters
for multiplexing. PCR products were gel-purified using a DNA extraction kit, and DNA
concentrations were measured using a Qubit fluorometer. The resulting libraries were submitted
to GENEWIZ for Illumina sequencing or AmpSeq using the Element AVITI sequencing platform,
which yields estimated per-base error probabilities of <0.1% per nucleotide. NGS data were first
demultiplexed, and variants at each position were extracted using cutadapt
(doi.org/10.14806/ej.17.1.200). The frequency of each amino acid was then calculated using a
custom R script. HIV-1 and SIVcpz sequences (n = 12,261) were obtained from the Los Alamos
HIV database.

For CA sequence analysis of adapted variants, PCR-amplified products covering the full coding
sequence of HIV-1 CA were submitted for Oxford Nanopore sequencing at Plasmidsaurus, which
is characterized by raw single-read per-base error probabilities of approximately 1–5% per
nucleotide. Sequencing reads were mapped to the reference sequence (the LAI strain) using
Hisat266 and inspected using IGV software67. The frequency of each mutation was extracted from
BAM files using a custom shell script. Mutation calls were verified by manual inspection in IGV. A
threshold of 5% was arbitrarily used to identify de novo mutations. For further analysis, we
included mutations exhibiting a significant frequency increase. Amino acid substitutions were
visualized on the CA hexamer structure (PDB: 3J34 and PDB: 6VKV) using UCSF ChimeraX68.

For the linkage analysis, cutadapt was used to remove sequences shorter than 50 bp and those
with a Phred quality score lower than 30. Burrows-Wheeler Aligner69 was used for alignment and
Samtools70 for indexing. Single nucleotide variants (SNVs) were called using LoFreq71, and
bcftools70 filtered out SNVs with a frequency less than 1% and those with Phred-scaled quality
values less than 500. A python script incorporating pysam70 and vcfpy (DOI:
10.21105/joss.00085) was written in-house and used to parse through the selected reads for the
selected SNVs. Reads that did not contain information for one or more of the SNVs were removed
from the data set. The output contained groups of mutations and their number of occurrences in
the remaining reads. This was manually manipulated in Microsoft Excel to remove any group of
SNVs with a frequency of less than 1%. These low-frequency groups of SNVs for each time point
were moved into the “Other” category for that time point. Once all linkage information was
determined, any group of SNVs which never rose above 5% at any time point was also moved to
the “Other” category for its respective time point.

Isolation and disassembly of HIV-1 cores. HIV-1 cores were obtained by ultracentrifuging intact
virus particles through a detergent layer onto a 30% sucrose cushion48. Specifically, 250 µL of 10-
fold concentrated viral stocks were subjected to ultracentrifugation at 4°C for 2 hours using a
Beckman SW50.1 rotor at 127,000 × g. The detergent layer contained 0.03% IGEPAL CO-630 in
a Tris-based buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA; STE buffer). The
isolated cores were then resuspended in the STE buffer for subsequent analysis.

To examine core disassembly, 50 µL of purified cores were incubated with or without 100 µM IP6
for 2 hours at either 4°C or 37°C. After incubation, core samples underwent microcentrifugation
at 20,817 × g for 10 minutes at 4°C. The resulting pellets were resuspended in 50 µL of RPMI
1640 medium supplemented with 10% FBS. A quantitative reverse transcriptase (RT) assay
described below was then performed on both isolated viral cores and the pellet samples after
incubation to evaluate core yield and disassembly. To determine the effects of LEN on viral cores,
50 µL of purified cores were incubated with LEN at final concentrations of 0.004 µM, 0.02 µM, 0.1
µM, and 0.5 µM at 4°C for 1 hour. After the 1-hour incubation, the core samples were subjected
to a similar microcentrifugation process to separate the pelletable cores.

Quantitative reverse transcriptase assay. To quantify the amount of reverse transcriptase (RT),
a product-enhanced RT assay utilizing quantitative PCR (qPCR) was performed72. In this assay,
5 µL of input materials (such as virus stocks or isolated cores) was mixed with 5 µL of 2× lysis
buffer (40% glycerol, 0.25% Triton X-100, 100 mM Tris-HCl pH 7.5, and 50 mM KCl) in a 96-well
round-bottom plate. The mixture was incubated at room temperature for 10 minutes to allow lysis.
A pre-determined RT standard curve was generated using a virus stock with a known level of RT.
To stop the lysis process, 90 µL of distilled deionized water was added.

The reaction mixture consisted of 10 µL of lysed supernatant and 10 µL of 2× reaction buffer,
which contained 10 mM MgCl , 1× core buffer (20 mM Tris-HCl pH 8.3, 20 mM KCl, and 5 mM
2
(NH
4
)
2
SO
4
), 4 µg/mL BSA, 0.4 mM dNTPs, 1 µM forward primer (5’-
TCCTGCTCAACTTCCTGTCGAG), 1 µM reverse primer (5’-
CACAGGTCAAACCTCCTAGGAATG), 0.04 µg MS2 RNA (Sigma), 1× SYBR™ Green I Nucleic
Acid Gel Stain (Thermo Fisher Scientific), 0.02 µL Rox reference dye (Thermo Fisher Scientific),
and 0.4 U RiboLock RNase Inhibitor. Before initiating the reaction, 2 units of Taq polymerase
were added.

Reverse transcription of MS2 RNA was followed by qPCR using a real-time PCR system. For
assays performed in a 384-well plate format, half of the reaction volume was used. The thermal
cycling conditions were as follows: reverse transcription at 42°C for 20 minutes, initial
denaturation at 95°C for 2 minutes, followed by 40 cycles of amplification with denaturation at
95°C for 5 seconds, annealing at 60°C for 5 seconds, extension at 72°C for 15 seconds, and a
final step at 80°C for 25 seconds.

Western blotting. Virus particle (2 µl) and core samples (18 µl) were lysed with 4X Bolt™ LDS
Sample Buffer (Invitrogen) and resolved on NuPAGE Bis-Tris 4–12% polyacrylamide gels
(Invitrogen). Electrophoresis was carried out at 100 V for 60 minutes in a mini-gel tank. Following
electrophoresis, proteins were transferred onto a nitrocellulose membrane using a wet transfer
system with 1× Bolt Transfer Buffer (containing 10% methanol). Membranes were blocked
overnight at 4°C. After blocking, membranes were washed three times for 5 minutes each in 1×
TBS-T (Tris-buffered saline with 0.1% Tween-20), then incubated with mouse monoclonal anti-
HIV-1 p24 antibody (clone 183-H12-5C, ARP-3537, BEI) at a 1:1000 dilution for 2 hours at room
temperature with gentle agitation in a shaker. After three additional washes in 1× TBS-T, the
membranes were incubated with an IRDye 488-conjugated anti-mouse secondary antibody (LI-
COR Biotechnology) diluted 1:10,000 in blocking buffer for 1 hour at room temperature. Final
three washes (3 times, 5 minutes each) were performed in 1x TBST. Blots were imaged using a
Licor Odyssey system, and p24 signal intensities were quantified. Uncropped and unprocessed
scans of the blots are provided in the Source Data file.

Software and data analysis. Data were collected using commercially available software,
including QuantStudio Design & Analysis Software, NovoExpress, LI-COR Image Studio, and
Molecular Devices SoftMax Pro. Data analysis was performed using commercially available and
open-source software together with custom scripts. Software used included GraphPad Prism,
FlowJo, bcftools, Biopython, Biostrings, bwa, cutadapt, LoFreq_Star, pandas, pysam, samtools,
stringr, and vcfpy.

Statistical analysis. Statistical analyses were conducted using Student’s t-test or ANOVA,
implemented in Prism software (GraphPad). Statistical significance was defined as p < 0.05. ****p
< 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Overall, important study that attempts to elucidate HIV-1 viral evolution with regards to development of resistance to a novel class of antiretroviral agents (small molecule capsid inhibitors with lenacapavir as first-in-class) in context of varying drug selection pressures and implications for viral fitness and infectivity. I like the efforts to assess how additional accrued mutations may modify or modulate viral fitness- viz exploring how there may be compensatory mutations as a viral evolutionary strategy to improve/regain fitness. this has important implications for and relevance for a drug that is already approved for HIV treatment and is likely to be deployed widely globally for HIV prevention in the coming months/years.\nmy\nIntroduction- the statement- \"two individuals who acquired HIV-1 during LEN-based PrEP carried the LEN-resistant N74D variant either through selective transmission of this variant or via de novo evolution.\" leaves room to suggest that it could have been transmitted resistance. this is inaccurate as studies have shown that N74D is a treatment emergent mutation that is unlikely to be circulating in the community given that lenacapavir has not been used outside the context of small clinical trials thus lending greatest likelihood that it was exposure emergent. see https://pubmed.ncbi.nlm.nih.gov/32154864/\nMethods-\nfocus on variant with Q67H and N74D, limits generalizability of tis study as there have been multiple patterns of resistance mutation emergence- singly and accrued in combination in clinical studies of LEN. thus observed evolution patterns described in the paper may have limited applicability. for example in the Capella study, there is heterogeneity in pattern of resistance mutation emergence: foe example, some individuals developed M66I/M first, and others had T107A/S- so there is no one clear pattern of mutation accrual. https://journals.sagepub.com/doi/full/10.1177/13596535231220754\n- while i understand dose escalation approaches are great to define viral evolution/adaptation in in-vitro models, in the clinical space, maybe except for a patient population such as in Capella where lenacapavir is continued because of limited treatment options even in the face of emergent resistance; the opposite scenario is likely to occur when used for HIv prevention where infections occur as drug levels wane/ fall below target levels, such that viral mutational \"escape\" in that context may behave differently- will like authors to address this point.\n- not much work on HIV-2 has been conducted in this space (susceptibility to and evolution of resistance to capsid inhibitors)- this has relevance for parts of the world where there are significant background rates of HIV-1/HIV-2 coinfection or HIV-2 monoinfection. experiments on HIV-2 will expand global relevance of this study\nDiscussion:\n- of course in-vitro systems may present different results than in-vivo experience, but how do authors explain detection of novel mutations in their in-vitro systems that have not been elicited in broad ranging preclinical and clinical development studies for lenacapavir or its precursor GS-CA-1 such as E187K that they characterize in so much detail?\nrug", "reviewer_2": "In this study, Orris et al. employed an accelerated evolution approach to study HIV’s resistance pathways to lenacapavir (LEN). Their key findings include (1) despite generally accepted sequence conservation in HIV capsid, the virus can develop novel and complex pathways to escape LEN effects, (2) M66I serves as a key bridge in resistance pathways, (3) a variant with combination of M66I+Q67H+E213K became LEN-dependent. Overall, this is a very well-written manuscript. Experimental methods and analyses were robustly performed. Results from the study provide important insights to our understanding of LEN resistance pathways.\nMajor comments.\n1. Clinical relevance of the author’s findings should be further clarified. For example, in their culture duration, what was the estimated rounds of replication were they expecting (given the known RT error rate in LAI). How does this compare to a viremic individual not on therapy? Essentially, what is the likelihood that one or more of these mutations would occur in a natural setting? Have any of these mutation and mutation combinations been observed in vivo without LEN pressure?\n2. One of the key findings is on drug concentration dependence. Since LEN is long acting, this study may suggest that individuals receiving LEN should be carefully monitored for suboptimal concentration – please include in the discussion. Is there a threshold that the authors would suggest?\nMinor comments.\nResults.\nLine 103-109 and Figure 1. Please clarify in-text: Was this done in the absence or presence of LEN?\nLine 127-128. Please clarify that the intolerance is within the context of subtype B LAI.\nLine 153-162 and Figure 2. Are you sure Fig 2a is Q67 and Fig 2b is N74? Both wildtypes were shown in the logo plot in 2b.\nLine 310. Is E187K naturally occurring in the absence of LEN?\nLine 414-416 and Figure 8c. It is unclear to me why the authors started the experiment with a strain containing N74D (in addition to Q67FHWY). Please clarify the rationale.\nDiscussion.\nObservations were based on LAI and subtype B, which should be discussed as a limitation in the discussion section.\nMethods.\nLine 801-802. Sequencing platform and error estimates not reported. Illumina, PacBio and Nanopore each have different baseline error rates.", "reviewer_3": "This manuscript represents a comprehensive assessment of the acquisition by HIV-1 of Lenacapavir resistance mutants in vitro. Its nicely done and well controlled. My comments are largely related to interpretation and can mostly be covered by text changes.\n1. Would have liked to have seen at least one experiment with a primary isolate of HIV-1. Would the mutations acquired be the same, particularly for a founder clone given LEN is used for prophylaxis.\n2. Should “in vitro” be included in the title to distinguish this work from in vivo studies?\n3. All LEN resistance was studied in the T-cell line MT4. My understanding is that this line is selected because its super permissive to HIV replication and particularly to HIV mutants that are defective in other cells, particularly primary cells. I guess this might skew results because maybe some mutations may only be replication competent in MT4. Or at least more replication competent in MT4. In this regard please address the following points in the text.\n3a Do we know if HIV-1 replication in MT4 selects change in the absence of inhibitor?\n3b What do we know about selection of resistance in vitro vs in vivo for other inhibitors, has this ever been compared. Eg does in vitro replication in the presence of eg integrase inhibitors or RT inhibitors select the same resistance profile. Does this support or undermine the present study.\n3c line 572, considering points above I don’t think sequence selection is an assay sensitivity issue its due to MT4 providing different selective pressure than in vivo replication. This point should be discussed.\n3d I think the authors should go beyond the statement on line 561 that the mutants may have a defect in primary cells, particularly since MT4 are the most permissive T-cell line. Can the authors test 1 or 2 key resistance mutants for replication in some primary cells? Do they get the same level of resistance/replication? Agreed, this would make the paper more compelling.\n4 line 562 I don’t think that there is an assumption that conserved viral sequences are inherently immutable. Most drug resistance studies undermine this notion."}, "author_response": "Response: We thank the reviewer for their careful evaluation of the manuscript and for the helpful comments provided. We appreciate that the reviewer found our study important. We believe that the revisions have improved the clarity and strength of the manuscript.\nIntroduction- the statement- \"two individuals who acquired HIV-1 during LEN-based PrEP carried the LEN-resistant N74D variant either through selective transmission of this variant or via de novo evolution.\" leaves room to suggest that it could have been transmitted resistance. this is inaccurate as studies have shown that N74D is a treatment emergent mutation that is unlikely to be circulating in the community given that lenacapavir has not been used outside the context of small clinical trials thus lending greatest likelihood that it was exposure emergent. see https://pubmed.ncbi.nlm.nih.gov/32154864/\nResponse: We agree. Although we initially considered both possibilities, as the reviewer correctly noted, this observation is most consistent with a de novo mutation. Accordingly, we have revised the sentence as follows:\n“Given that N74D is rarely observed among LEN-untreated HIV-1-infected individuals, this is most consistent with de novo evolution rather than selective transmission.”\nMethods-\nfocus on variant with Q67H and N74D, limits generalizability of tis study as there have been multiple patterns of resistance mutation emergence- singly and accrued in combination in clinical studies of LEN. thus observed evolution patterns described in the paper may have limited applicability. for example in the Capella study, there is heterogeneity in pattern of resistance mutation emergence: foe example, some individuals developed M66I/M first, and others had T107A/S- so there is no one clear pattern of mutation accrual.\nhttps://journals.sagepub.com/doi/full/10.1177/13596535231220754\nResponse: We appreciate the reviewer’s point regarding the heterogeneity of lenacapavir resistance pathways observed in clinical studies. While M66I has been reported as an early resistance mutation in studies such as CAPELLA, prior in vitro work has shown that M66I alone severely impairs viral infectivity and fails to acquire any secondary mutation under LEN pressure. Our study provides a mechanistic framework for how Q67H/N can facilitate the acquisition and tolerance of M66I, thereby defining plausible evolutionary trajectories that may occur in vivo. This is a point already discussed in the original manuscript (line 624). Notably, several of the mutational combinations examined here have also been observed in the CAPELLA study.\nWe agree that lenacapavir resistance can emerge through multiple heterogeneous pathways, which is further underscored by our new observation that the CH505 virus carrying Q67H adapted under lenacapavir pressure without acquiring M66I. While our study does not aim to capture the full spectrum of resistance patterns, it highlights specific, clinically relevant trajectories and points to important areas for future investigation.\n- while i understand dose escalation approaches are great to define viral evolution/adaptation in in-vitro models, in the clinical space, maybe except for a patient population such as in Capella where lenacapavir is continued because of limited treatment options even in the face of emergent resistance; the opposite scenario is likely to occur when used for HIv prevention where infections occur as drug levels wane/ fall below target levels, such that viral mutational \"escape\" in that context may behave differently- will like authors to address this point.\nResponse: This is a great point, and we agree that drug concentration is likely a key factor shaping viral evolution and adaptation. We have added the following description to the fifth paragraph of the Discussion:\n“This difference may reflect the use of a dose-escalation strategy in our adaptation scheme. If drug concentrations instead remain fixed or were changed only modestly in a manner that more closely mirrors pharmacokinetic dynamics in individuals receiving LEN, alternative resistance pathways may be selected. Indeed, the complex evolutionary patterns observed, including both M66I-containing mutational signatures and M66I-independent trajectories, may be driven by heterogeneity in drug exposure.”\n- not much work on HIV-2 has been conducted in this space (susceptibility to and evolution of resistance to capsid inhibitors)- this has relevance for parts of the world where there are significant background rates of HIV-1/HIV-2 coinfection or HIV-2 monoinfection. experiments on HIV-2 will expand global relevance of this study\nResponse: We agree that HIV-2 represents an important and understudied context for capsid inhibitor resistance with significant global relevance. The present study focuses on mechanistic and evolutionary analysis of lenacapavir resistance in HIV-1 using clinically observed mutations and established experimental systems. Extending these analyses to HIV-2 would require the development of replication-competent HIV-2 systems and dedicated evolutionary studies, which are beyond the scope of the present work.\nDiscussion:\n- of course in-vitro systems may present different results than in-vivo experience, but how do authors explain detection of novel mutations in their in-vitro systems that have not been elicited in broad ranging preclinical and clinical development studies for lenacapavir or its precursor GS-CA-1 such as E187K that they characterize in so much detail?\nResponse: In our experiments, E187K emerged under conditions of strong and sustained lenacapavir pressure, including dose-escalation and high drug concentrations. While physiologically active LEN levels can approach ~200 nM, sustained selection at such high exposure is likely to occur only in limited contexts, given the current patterns of LEN deployment and the rarity of functional LEN monotherapy in vivo. This may explain why E187K has not been detected in prior preclinical or clinical studies. We have added the following clarification to the eighth paragraph of the Discussion:\n“The absence of E187K in prior studies may reflect the rarity of sustained selection under high LEN exposure; although physiologically active LEN concentrations can approach ~200 nM, such levels are unlikely to be commonly or persistently achieved in vivo.”\nIn addition, characterization of E187K and other CA-CTD mutations (such as E213K) was motivated by a mechanistic interest in understanding how the capsid lattice can further adapt under strong selective pressure, providing fundamental insight into the constraints and plasticity of the HIV-1 capsid.\nReviewer #2\nIn this study, Orris et al. employed an accelerated evolution approach to study HIV’s resistance pathways to lenacapavir (LEN). Their key findings include (1) despite generally accepted sequence conservation in HIV capsid, the virus can develop novel and complex pathways to escape LEN effects, (2) M66I serves as a key bridge in resistance pathways, (3) a variant with combination of M66I+Q67H+E213K became LEN-dependent. Overall, this is a very well-written manuscript. Experimental methods and analyses were robustly performed. Results from the study provide important insights to our understanding of LEN resistance pathways.\nResponse: The reviewer provides an overall positive assessment of the manuscript (noting that it is well-written, robustly performed, and provides important insights). We appreciate these comments and have revised the manuscript accordingly, addressing each point in detail below.\nMajor comments.\n1. Clinical relevance of the author’s findings should be further clarified. For example, in their culture duration, what was the estimated rounds of replication were they expecting (given the known RT error rate in LAI). How does this compare to a viremic individual not on therapy? Essentially, what is the likelihood that one or more of these mutations would occur in a natural setting? Have any of these mutation and mutation combinations been observed in vivo without LEN pressure?\nResponse: We thank the reviewer for raising the question of clinical relevance and the likelihood that the identified mutations could arise in a natural setting. Rather than introducing a separate discussion section, we addressed this point within the existing Discussion paragraph focused on evolutionary trajectories involving M66I, as this context best reflects our interpretation of the data.\nGiven the high mutational rate of HIV-1 (approximately one genome in three acquiring at least one mutation per replication cycle) and the extraordinarily large number of virions produced daily in untreated infection, the mutational supply in vivo is orders of magnitude greater than that typically achieved in vitro, even in large-scale culture systems. Consequently, single-nucleotide substitutions such as Q67H or M66I are expected to arise stochastically in vivo. However, in the absence of LEN pressure, such variants are unlikely to be observed, which would be constrained by competition with WT virus and associated fitness costs.\nAlthough double mutants could, in principle, be generated at very low frequency given the large mutational supply in vivo, their persistence and evolutionary impact in drug-free conditions would be expected to be rare, particularly for combinations involving M66I, which is strongly fitness-impaired on its own. Accordingly, rather than invoking selection of rare pre-existing double mutants, our data support a stepwise evolutionary model in which compensatory substitutions such as Q67H or A105T enable the emergence and maintenance of highly resistant M66I-containing variants specifically under LEN pressure.\nWe have clarified this interpretation in the 6th paragraph of the Discussion section.\n“Notably, substitutions such as M66I are not frequently observed in the absence of LEN pressure. However, given the high mutational supply generated during untreated HIV-1 replication, single-nucleotide substitutions are expected to arise stochastically in vivo but are unlikely to be detected unless they reach sufficient frequency, which would be constrained by fitness and competition with WT virus. While double mutants could in principle be generated at very low frequency, their persistence in the absence of selection would be expected to be rare due to associated fitness costs.”\n2. One of the key findings is on drug concentration dependence. Since LEN is long acting, this study may suggest that individuals receiving LEN should be carefully monitored for suboptimal concentration – please include in the discussion. Is there a threshold that the authors would suggest?\nResponse: We appreciate the reviewer’s comment regarding drug concentration dependence in the context of long-acting LEN. Our study does not address pharmacokinetic thresholds, and the LEN-dependent variants described here emerged only under high LEN concentrations exceeding those used clinically. As such, we do not believe our data support proposing concentration-based monitoring strategies or defining a specific threshold. Instead, and consistent with the emphasis already made in the Discussion, we consider virologic and genetic monitoring in the setting of breakthrough infection to be the most informative and practical approach for identifying and interpreting such rare phenotypes. For this reason, we did not expand the main text further beyond the existing discussion.\nMinor comments.\nResults.\nLine 103-109 and Figure 1. Please clarify in-text: Was this done in the absence or presence of LEN?\nResponse: We thank the reviewer for pointing out this ambiguity. The experiment shown Figure 1 was performed in the absence of LEN. We have clarified this by adding “in the absence of LEN” to the main text (Results section, second paragraph) and to the legend of Figure 1.\nLine 127-128. Please clarify that the intolerance is within the context of subtype B LAI.\nResponse: We agree with the reviewer. We have explicitly mentioned that this observation was made with the LAI backbone. has now been modified as follows:\n“Sequence analysis of variants selected five days post-expansion revealed highly variable degrees of mutational tolerance in these four positions, with an unexpected diversity of viable variants found in three of the four mutant libraries using an LAI backbone.”\nAdditionally, this point is briefly revisited in the Discussion of the revised manuscript in light of our new results using the subtype C CH505 capsid; further discussion is provided below.\nLine 153-162 and Figure 2. Are you sure Fig 2a is Q67 and Fig 2b is N74? Both wildtypes were shown in the logo plot in 2b.\nResponse: We apologize for this oversight and thank the reviewer for drawing our attention to it. The figures have been corrected such that Figure 2a shows N74 and Figure 2b shows Q67.\nLine 310. Is E187K naturally occurring in the absence of LEN?\nResponses: E187K appears to be extremely rare in the absence of lenacapavir (LEN) selection. We surveyed approximately 8,000 HIV-1 p24 (capsid) sequences from the Los Alamos HIV Sequence Database and identified E187K in 5 out of 8,003 sequences (<0.1%). Given this very low frequency, it is difficult to determine whether these represent bona fide naturally occurring variants or reflect sequencing errors or annotation artifacts. Overall, these data indicate that E187K is not a common naturally occurring polymorphism in untreated HIV-1 populations.\nLine 414-416 and Figure 8c. It is unclear to me why the authors started the experiment with a strain containing N74D (in addition to Q67FHWY). Please clarify the rationale.\nResponse: We apologize for the lack of clarity. The experiment was not initiated with an N74D-containing virus. Selection was started from the M66I+Q67H background, and N74D arose de novo under lenacapavir pressure and became nearly fixed at 200 nM. Upon further escalation of drug concentration to 400 nM, an additional mutation, A77V, was acquired on the M66I+Q67H+N74D background, resulting in the quadruple mutant shown in Figure 8c. We have revised the text to explicitly clarify the sequential emergence of these mutations.\n“Both substitutions reside within the drug-binding site and were maintained at higher drug concentrations. A notable de novo mutation that emerged during selection of the M66I+Q67H background was the well-known LEN resistance mutation N74D, which became nearly fixed at 200 nM; continued selection at 400 nM subsequently led to acquisition of A77V, resulting in the M66I+Q67H+N74D+A77V genotype.”\nDiscussion.\nObservations were based on LAI and subtype B, which should be discussed as a limitation in the discussion section.\nResponse: We agree with the reviewer. The initial experiments were performed using LAI, a subtype B virus, and we now explicitly discuss this as a limitation in the Discussion. In addition, prompted by this comment and a related comment from another reviewer, we performed dose-escalation adaptation experiments using a virus carrying the CH505 capsid (subtype C), starting from the Q67H background. Notably, the adaptation pattern observed with CH505 CA differed from that observed with LAI CA; in contrast to the emergence of M66I in the LAI background, CH505 CA selected for a distinct set of mutations, which are shown in Figure S14. Together, the limitation along with these new findings and their implications for subtype- and background-dependent adaptation are now discussed in the revised Discussion as follows:\n“Finally, our mutagenesis work solely relied on the LAI CA backbone. Hence, the mutational tolerance we observed may not be universal across other strains or subtypes. Recent work, however, has shown that the pattern of early-emerging LEN resistance mutations is highly similar across different subtypes. In contrast, our in vitro adaptation using a virus harboring the capsid with a Q67H substitution from a T/F strain of HIV-1 (CH505) displayed a distinct evolutionary trajectory from that observed in our LAI-based adaptation, which recurrently selected for the strong M66I substitution. Thus, while the mechanistic basis for these differential evolutionary pathways remains to be defined, these results highlight the importance of capsid sequence context in LEN resistance evolution.”\nMethods.\nLine 801-802. Sequencing platform and error estimates not reported. Illumina, PacBio and Nanopore each have different baseline error rates.\nThank you for pointing out this oversight in our manuscript. In the revised manuscript, information on the sequencing platforms has been added as follows.\n“The resulting libraries were submitted to GENEWIZ for Illumina sequencing or AmpSeq using the Element AVITI sequencing platform, which yields estimated per-base error probabilities of <0.1% per nucleotide.”\n“For CA sequence analysis of adapted variants, PCR-amplified products covering the full coding sequence of HIV-1 CA were submitted for Oxford Nanopore sequencing at Plasmidsaurus, which is characterized by raw single-read per-base error probabilities of approximately 1–5% per nucleotide.”\nReviewer #3\nThis manuscript represents a comprehensive assessment of the acquisition by HIV-1 of Lenacapavir resistance mutants in vitro. Its nicely done and well controlled. My comments are largely related to interpretation and can mostly be covered by text changes.\nResponse: The reviewer’s comments were helpful in improving the manuscript. It should be also noted that the positive evaluation of the work as “nicely done and well controlled” is appreciated. All points raised have been addressed in the responses below.\n1. Would have liked to have seen at least one experiment with a primary isolate of HIV-1. Would the mutations acquired be the same, particularly for a founder clone given LEN is used for prophylaxis.\nResponse: We agree with the reviewer that evaluating resistance evolution in a primary isolate or transmitted/founder (TF) context is important, particularly given the use of lenacapavir (LEN) in prophylaxis settings. After submission of our manuscript, Naik et al. published a study examining resistance mutations across a diverse collection of HIV-1 strains and found that resistance profiles are largely conserved across HIV-1 subtypes (PMID: 40965168).\nThus, this question has now been addressed independently; however, we also became interested in exploring evolutionary pathways beyond the initial amino acid substitution. Accordingly, we performed an additional dose-escalation adaptation experiment using a newly generated LAI-based chimeric virus carrying the capsid (CA) sequence from the clade C TF virus CH505 which we then added Q67H substitution to.\nThis virus was successfully adapted to higher LEN concentrations, demonstrating that LEN resistance evolution is not restricted to the LAI CA background. During adaptation, we identified the emergence of a K70R substitution. Notably, the Q67H+K70R combination has been observed in the CAPELLA clinical study, further supporting the recurrent evolutionary patterns emphasized in this manuscript. With continued adaptation, an additional substitution (A174T) emerged, indicating engagement of both recurrent and novel evolutionary pathways.\nImportantly, this adaptation trajectory differed from the LAI-based pathway characterized by acquisition of M66I, suggesting that capsid genetic background influences accessible LEN resistance routes. While defining the precise determinants underlying these background-dependent differences is beyond the scope of the current study, the emergence of clinically observed substitutions (Q67H+K70R) in a transmitted/founder–derived capsid context directly addresses concerns raised by Reviewer 2 regarding the clinical relevance of laboratory strain-based adaptation. Together, these data strengthen the clinical relevance of our findings and support the conclusion that both recurrent and context-dependent evolutionary pathways can contribute to LEN resistance.\nThis has been added to the last paragraph of the Results section and discussed in the paragraph before the final paragraph of the Discussion section. Given that these changes span multiple paragraphs, we refer the reviewer to the indicated sections of the revised manuscript.\n2. Should “in vitro” be included in the title to distinguish this work from in vivo studies?\nResponse: We agree with the reviewer. To clearly distinguish this work from in vivo studies, we have revised the manuscript title to include “in vitro” as follows:\n“Recurrent and Novel Evolutionary Pathways Drive in vitro HIV-1 Lenacapavir Resistance with Diverse Phenotypic Consequences.”\n3. All LEN resistance was studied in the T-cell line MT4. My understanding is that this line is selected because its super permissive to HIV replication and particularly to HIV mutants that are defective in other cells, particularly primary cells. I guess this might skew results because maybe some mutations may only be replication competent in MT4. Or at least more replication competent in MT4. In this regard please address the following points in the text.\n3a Do we know if HIV-1 replication in MT4 selects change in the absence of inhibitor?\nWhat was the long-term evolution study?\nResponse: Yes, long-term passaging of HIV-1 in MT-4 cells in the absence of antiviral drugs can select for adaptive changes related to growth in this cellular environment, as shown in prior experimental evolution studies (e.g., Bertels et al.: PMID: 31251344). However, in our study, the mutations analyzed were selected under lenacapavir pressure, and most of the resistance-associated substitutions we tested either increased viral fitness or conferred resistance rather than reflecting general adaptation to MT-4 cells. Accordingly, we do not believe that MT-4-specific adaptation is a major driver of the resistance pathways described here.\n3b What do we know about selection of resistance in vitro vs in vivo for other inhibitors, has this ever been compared. Eg does in vitro replication in the presence of eg integrase inhibitors or RT inhibitors select the same resistance profile. Does this support or undermine the present study.\nResponse: Both outcomes have been reported. For multiple antiretroviral classes, including reverse transcriptase and integrase inhibitors, most resistance mutations identified through in vitro selection are also observed in vivo, and many have been validated clinically. However, certain resistance mutations that arise in vivo require multi-nucleotide changes and are therefore less readily accessed by standard in vitro selection approaches.\nThis distinction was a key motivation for our experimental design. As noted in the 1st paragraph of the Results section, we used site-saturation mutagenesis to systematically probe less-accessible, multi-nucleotide drug-resistance mutations that frequently emerge in clinical settings but may be underrepresented in conventional in vitro selection experiments. Thus, rather than undermining our conclusions, prior comparisons between in vitro and in vivo resistance evolution support the rationale and relevance of the present study.\n3c line 572, considering points above I don’t think sequence selection is an assay sensitivity issue its due to MT4 providing different selective pressure than in vivo replication. This point should be discussed.\nResponse: We agree that differences in cellular environment can contribute to differences in selective pressure between in vitro and in vivo settings. Additionally, we realized that N74S emerged in one of the Q67N dose escalation experiments (Supplementary Fig. 6). To clarify this point, while incorporating the reviewer’s comment and our discovery, we have revised the sentence as follows:\n“Our inability to detect N74S, which appeared alongside Q67H in one LEN-treated individual, may be due to a potential auxiliary role in supporting DRMs at position 67, differences in drug pressure and/or cellular environment between in vitro and in vivo settings.”\n3d I think the authors should go beyond the statement on line 561 that the mutants may have a defect in primary cells, particularly since MT4 are the most permissive T-cell line. Can the authors test 1 or 2 key resistance mutants for replication in some primary cells? Do they get the same level of resistance/replication? Agreed, this would make the paper more compelling.\nResponse: We agree that directly assessing replication competence in primary CD4⁺ T cells is important given the high permissiveness of MT-4 cells. In response to this comment, we examined the infectivity and spreading capacity of representative lenacapavir-resistant variants in primary CD4⁺ T cells. This is now shown in Fig. S5 and mentioned in text as follows:\n“We next assessed the viral infectivity and replicative capacity of Q67H and Q67N in primary CD4⁺ T cells. Single-round infectivity was comparable between Q67H and Q67N, while Q67H showed a modest trend toward higher replicative capacity relative to Q67N that did not reach statistical significance. As controls, M66I and N74D exhibited markedly impaired infectivity and replication in primary CD4⁺ T cells (Supplementary Fig. 5).”\nAs expected, the highly resistant M66I mutant exhibited severely impaired infectivity, whereas Q67H and Q67N retained infectivity and spreading kinetics comparable to wild-type virus. In contrast, the N74D variant showed impaired replication in primary CD4⁺ T cells, consistent with our prior observations and confirmed here. Together, these results indicate that, for most representative variants, replication competence in primary CD4⁺ T cells parallels that observed in MT-4 cells.\nThis is now described in the revised manuscript as follows:\n“We next assessed the viral infectivity and replicative capacity of Q67H and Q67N in primary CD4⁺ T cells. Single-round infectivity was comparable between Q67H and Q67N, while Q67H showed a modest trend toward higher replicative capacity relative to Q67N that did not reach statistical significance. As controls, M66I and N74D exhibited markedly impaired infectivity and replication in primary CD4⁺ T cells (Supplementary Fig. 5).”\nThe sentence in the Discussion section has been changed as follows:\n“It is also possible that some viable variants identified in this study have a defect in replicating in primary CD4+ T cells, although limited analyses of HIV-1 mutants in primary CD4⁺ T cells show infectivity and replicative properties similar to those in MT-4 cells.”\nWe note that these experiments were designed to address replication and infectivity, which was the primary concern raised here. A systematic evaluation of drug resistance levels in primary cells would require additional assay optimization and we believe this point falls outside the focus of the current work.\n4 line 562 I don’t think that there is an assumption that conserved viral sequences are inherently immutable. Most drug resistance studies undermine this notion.\nResponse: We agree that “inherently immutable” overstates the prevailing view. We have therefore revised the sentence to adopt more precise language, emphasizing evolutionary constraint rather than immutability. The revised text now reads:\n“Nonetheless, our data challenge the view that highly conserved viral sequences are strictly constrained and demonstrate that even evolutionarily conserved regions can accommodate resistance-associated variation under strong selective pressure.”\nResponse: This is an important question. Our answer is not necessarily. Replicative capacity and transmission fitness are related but distinct properties, and resistance mutations that replicate efficiently in vitro may not transmit with equal efficiency in vivo. Transmission is influenced by additional selective pressures present at the mucosal barrier and during early infection, including innate immune responses.\nIn this context, we note that certain lenacapavir-resistant variants (e.g., N74D) show increased sensitivity to type I interferon, whereas interferon resistance is a characteristic frequently associated with transmitted founder viruses. This raises the possibility that some resistance mutations may carry a transmission cost even if they retain substantial replicative capacity. While our study was not designed to directly test transmission fitness, this distinction is particularly relevant for considering LEN in prophylaxis settings and represents an important area for future investigation.\nResponse: We agree that interactions between the capsid lattice and FG-containing nucleoporins, including Nup153, play an important role in HIV-1 nuclear entry and that LEN binding could influence these interactions. However, prior work (PMID: 40231850) has shown that M66I, which impairs infectivity in non-dividing cells, does retain Nup153 binding, indicating that additional capsid-dependent properties likely contribute to this phenotype. Given this complexity, we have chosen not to further elaborate on specific FG-nucleoporin binding models\nResponse: We agree that, for some capsid-targeting compounds (e.g., PF74), dose-response curve shapes can provide mechanistic insight, and we have previously shown concentration-dependent effects using this approach. However, in our hands, lenacapavir exhibits extremely potent antiviral activity, and we did not observe distinct or multi-phase dose-response behaviors that could be readily interpreted as reflecting separable modes of inhibition. In addition, once highly resistant variants such as M66I emerge, the level of resistance is so pronounced that it becomes difficult to meaningfully resolve concentration-dependent effects based on dose-response curve shape alone. Accordingly, while the reviewer’s hypothesis is plausible, our data do not reveal clear evidence for distinct resistance profiles linked to different LEN concentration-dependent modes of action.\nResponse: We agree with the reviewer and have removed all instances of “data not shown” from the manuscript. Two such cases were present in the original submission, and both have now been addressed.\nFirst, we removed the clause referencing unpublished data and revised the text to link directly to the preceding observations. The revised sentence now reads:\n“Various E213K-containing molecular clones were constructed but found to be non-infectious. As these E213K variants emerged upon replication in high LEN concentrations, we hypothesized that variants carrying E213K may depend on LEN for infection.”\nSecond, the result showing that the N183K mutation alone does not confer LEN resistance has now been included in Supplementary Fig. 9 of the revised manuscript.\n“Interestingly, N183K, which emerged after high-dose selection and resides close to E187K, also did not affect LEN sensitivity (Supplementary Fig. 9).”\nResponse: We agree that this is an important conceptual point. We now discuss that hexamer stabilization can be accompanied by hexamer flattening, which introduces lattice-level strain and can destabilize the assembled viral core, as described previously. This clarification helps resolve the apparent paradox between increased core yield and LEN’s core-disrupting activity, while remaining consistent with prior observations linking increased core stabilization to enhanced LEN resistance. The revised part is as follows:\n“One possible explanation is that stabilization of individual CA hexamers can be accompanied by hexamer flattening, which introduces lattice-level strain and destabilizes the assembled core. Consistent with this view, previous work showed that increased core stabilization, either through mutations or IP6, is associated with enhanced LEN resistance.”\nResponse: Apologies for this oversight, and we thank the reviewer for catching it. The second abstract was an outdated version and has been removed from the revised manuscript.\nResponse: We thank the reviewer for this suggestion. The number of HIV-1/SIVcpz sequences used to generate Figure 1b (12,261) has now been added to the figure legend.\nResponse: We thank the reviewer for catching this error. The labeling in the Figure 2 legend has been corrected.\nResponse: In response to this comment, we performed a replicate experiment for the Q67 library. These data have now been added to Supplementary Figure 2. As shown, there is a strong correlation between the first and second experiments, demonstrating reproducibility.\nResponse: We thank the reviewer for this suggestion. In response, we have added infectivity measurements relative to wild-type virus in the absence of LEN for the cloned variants shown in Figure 6c (see below), demonstrating that acquisition of LEN resistance is accompanied by a reduction in viral fitness.\nIn contrast, performing an equivalent analysis for Figure 3b is not straightforward, as these samples represent adapted viral populations propagated in the presence of LEN and contain heterogeneous viral material with variable residual drug exposure. Normalizing infectivity in this context would therefore be difficult to interpret. Importantly, the data shown in Figure 6a–c, using molecularly cloned variants, clearly demonstrate a progressive loss of viral infectivity as higher levels of LEN resistance are acquired.\nResponse: We thank the reviewer for this helpful suggestion. In response, we have generated a new supplementary figure (now Figure S8) in which the relevant residues are mapped onto a capsid structure with LEN bound (PDB 6VKV), illustrating their spatial relationship to the LEN-binding site and supporting the statement in line 316.\nResponse: The following information has been added to the relevant figure legends.\nFigure 7c. Left panel: N = 6. Right panel: WT (N = 12), M66I+Q67N (N = 9), and M66I+Q67N+E187K (N = 12)\nFigure 7e. Left panel: N = 7. Right panel: WT (N = 6), M66I+Q67N (N = 4), and M66I+Q67N+E187K (N = 6)\nFigure 7g. N = 3\nFigure 9b. WT (N = 8), Q67N (N = 6), M66I+Q67N (N = 4), and M66I+Q67N+E187K (N = 4).\nFigure 9d. N = 4\nIn addition, upon re-examining the dataset, we noted that the assignment of P value (*) for comparing M66I+Q67N+E187K to WT in the right panel of Figure 7C was incorrect, which has now been assigned to **. The revised analysis has been incorporated into the manuscript.\nResponse: The “ns” annotation does refer to the comparison between WT and M66I/Q67N. Although a visual trend is apparent, this comparison did not reach statistical significance.\nResponse: We agree. Accordingly, the sentence has been modified as follows:\n“As expected from the known core-disrupting activity of lenacapavir27,28, LEN treatment resulted in a steady decrease in pelletable cores of the WT virus up to 20 nM, at which the curve plateaued (Fig. 7g).”\nResponse: We agree and have added a brief discussion citing prior work linking residues at the trimeric interface to Nup153 binding and nuclear import (see below). As nuclear entry was not directly examined in this study, we have confined our interpretation in this section to effects on capsid stability.\n“Although not addressed here, prior work has shown that residues at the trimeric interface, including E75 and E213, can interact with the RRR motif of Nup153, suggesting that this region may also contribute to nuclear import.”\nFinally, we made minor editorial revisions throughout the manuscript to correct grammatical errors. This included small refinements in terminology, primarily related to the use of the term “mutation” (e.g., replacing “mutations in the capsid protein” with “capsid mutations” for consistency and precision). As these changes are distributed throughout the manuscript and do not affect the substance of the work, they are not listed individually but are highlighted in blue"}, {"round": 2, "reviewer_comments": {"reviewer_2": "I have no additional comments. The authors have fully addressed all of my concerns. This is a very well-designed study and a well-written manuscript.", "reviewer_3": "The authors have addressed the points I made effectively. I have no further comments on the manuscript"}, "author_response": ""}]

Decision letter