Initiation of HIV-1 Gag lattice assembly is required for cytoplasmic recognition of the viral genome packaging signal

The encapsidation of HIV-1 genomic RNA into virions is enabled by the binding of the nucleocapsid (NC) domain of the HIV-1 Gag polyprotein to the structured viral RNA packaging signal (Ψ) at the 5’ end of the viral genome. However, the subcellular location and oligomeric status of Gag during the initial Gag-Ψ encounter remains uncertain. Domains other than NC, such as capsid (CA), may therefore indirectly affect RNA recognition. To investigate the contribution of Gag domains to Ψ recognition in a cellular environment, we performed protein-protein crosslinking and protein-RNA crosslinking immunoprecipitation coupled with sequencing (CLIP-seq) experiments. We demonstrate that NC alone does not bind specifically to Ψ in living cells, whereas full-length Gag and a CANC subdomain bind to Ψ with high specificity. Perturbation of the Ψ RNA structure or NC zinc fingers affected CANC:Ψ binding specificity. Notably, CANC variants with substitutions that disrupt CA:CA dimer, trimer or hexamer interfaces in the immature Gag lattice also affected RNA binding, and mutants that were unable to assemble a nascent Gag lattice completely lost the ability to specific bind to Ψ. Artificially multimerized NC domains did not specifically bind Ψ. CA variants with substitutions in inositol phosphate coordinating residues that prevent CA hexamerization were also deficient in Ψ binding and second-site revertant mutants that restored CA assembly also restored specific binding to Ψ. Overall, these data indicate that the correct assembly of a nascent immature CA lattice is required for the specific interaction between Gag and Ψ in cells.


Introduction
The generation of infectious HIV-1 virions depends on the encapsidation of two copies of HIV-1 genomic RNA (gRNA) into virions. HIV-1 gRNA is present among a vast excess of host RNAs in the cytosol of infected cells, yet it is the dominant RNA species in virions (Rulli et al., 2007). The specific encapsidation of HIV-1 gRNA into virions is attributed to the specific interaction between HIV-1 Gag and the viral packaging signal "Ψ" (Aldovini and Young, 1990;Berkowitz et al., 1996;Clavel and Orenstein, 1990;Clever et al., 1995Clever et al., , 2000Darlix et al., 1990;D'Souza and Summers, 2005;Lever et al., 1989). The Gag protein consists of three major domains: matrix (MA), capsid (CA), and nucleocapsid (NC). The NC domain binds directly to RNA, and point mutations or deletions disrupting the zinc fingers of NC interfere with the specific packaging of gRNA (Aldovini and Young, 1990;Gorelick et al., 1990). Since the NC domain is present in the context of Gag precursor when initiating interactions with Ψ, other domains of Gag may also contribute to Ψ binding specificity. Indeed, RNase protection assay, yeast-three hybrid assays, and in-vitro binding assays suggest that full-length Gag or CANC has different RNA binding specificity compared to NC and some studies indicate binding to Ψ with higher specificity than NC (Bacharach and Goff, 1998;Damgaard et al., 1998;Guo et al., 2020;Kroupa et al., 2020;Webb et al., 2013). Moreover, Gag mutants with CA mutations exhibited reduced gRNA selectivity in an in-vitro reconstituted HIV-1 Ψ packaging system (Carlson et al., 2016), while Gag mutants with CA assembly deficits cannot initiate gRNA packaging (Duchon et al., 2021;Kutluay et al., 2014;Kutluay and Bieniasz, 2010).
Nevertheless, there remains significant uncertainty as to the location, stoichiometry, and the overall nature of the protein RNA complex that initiates HIV-1 assembly. To probe the potential role of CA and early assembly events in the specific interaction between Gag and Ψ in biologically relevant settings, we performed CLIP experiments to determine whether fulllength Gag, a CANC subdomain, or NC alone could specifically bind to Ψ within HIV-1 gRNA in cells. We found that full-length Gag and CANC both bind to Ψ with high specificity in the cytosol, whereas an isolated NC domain does not. Through further studies of CA mutants, we demonstrate that manipulations which perturb any of the CA:CA interfaces required for the formation of an immature Gag lattice also perturb the specific interaction between Gag/CANC and Ψ. Our findings suggest that assembly of a nascent immature Gag lattice is required for the initial recognition of Ψ and the initiation of viral RNA packaging in the infected cell cytoplasm.

CLIP method for assessing Gag-RNA interaction in cells
We used a modified CLIP method based on published protocols (Kutluay et al., 2014;Shema Mugisha et al., 2020) to measure HIV-1 Gag-RNA binding interactions in living cells. In this CLIP procedure, an infrared-dye-conjugated 3' adaptor (Zarnegar et al., 2016) is used in place of a conventional radioactive labeled 3' adaptor. Protein-RNA crosslinking is driven by metabolic 4-thiouridine incorporation into target RNA (Hafner et al., 2010)  For CLIP experiments, we used derivatives of an HIV-1NL4-3 based proviral construct, referred to as HIV-1NL4-3 (MA-3xHA/PR -) that carries a 3xHA epitope tag, between the MA and CA domains of Gag, and encodes an inactivated protease (Kutluay et al., 2014), ( Figure 1A).
As we intended to study the initial Gag:gRNA interaction in the cytosol, and MA is not involved in this process (Bou-Nader et al., 2021;Kutluay et al., 2014), we also generated a proviral construct (referred to as pCANC) in which the start codon of MA (ATG) was mutated to abolish MA expression; instead, an N-terminally 3xHA CANC Gag fragment, along with the C-terminal SP2-p6 extension is expressed ( Figure 1A). Crucially, the RNA binding profile of the CANC protein to the HIV-1 genome was similar to that of the full-length Gag protein, with a marginally higher fraction of reads mapping to Ψ. We speculate that this apparently enhanced preference of CANC for Ψ is because the membrane bound Gag (that exhibits more promiscuous binding to the viral genome) (Kutluay et al., 2014) is not present when CANC is used.
Both the full length Gag protein and CANC exhibited characteristic binding to Ψ involving 3 'peaks' of read intensity corresponding to U5, SL1 and SL3/4 that are brought into proximity in the folded minimal packaging structure (Bieniasz and Telesnitsky, 2018;Lu et al., 2011) (Figure 1 B, C). To investigate the effects of RNA perturbation on CANC recognition, we prepared two Ψ mutants. In one mutant the GC-rich 'kissing' loop (GCGCGC) in the dimerization initiation site (DIS) was substituted with a GAGA tetraloop (CANC DIS-GAGA) top prevent RNA dimerization (Lu et al., 2011). In a second mutant, four bases "GGAA" were appended at the 5' end of the gRNA, immediately 5' to three guanosines at the transcription start site of HIV-1 viral genome (CANC InsGGAA). These nucleotides, along with the 5'cap affect the structure of the entire 5' leader (Ding et al., 2021). While CANC bound to the unmanipulated Ψ with high specificity, the CANC InsGGAA Ψ mutant was bound comparatively poorly by CANC, with failure of the 3' portion of Ψ to bind CANC ( Figure 1B, C). This finding is consistent with previous studies showing that the exposure of the 5'-cap of gRNA to the translational machinery due to "GGAA'' insertion inhibits gRNA packaging (Ding et al., 2021). The CANC DIS-GAGA Ψ mutant, which presumably favors the retention of a monomeric RNA was bound by CANC specifically with the Ψ element, but with the exclusion of the SL1 stem-loop. This finding is consistent with previous findings that other Ψ elements are retained in the DIS-GAGA structure as in native dimer (Keane et al., 2015), and suggests Figure 1. CLIP method validation and effects of Ψ perturbations on Gag recognition (A) Schematic representation of the pCANC construct. Asterisk indicates the mutation introduced at the MA start codon (ATG to ACG). (B) Read density distribution on viral RNA from CLIP experiments in which constructs encoding Gag, CANC, or CANC with mutant Ψ elements (CANC DIS-GAGA, and CANC InsGGAA) were used. The Yaxis represents the decimal fraction of all reads that mapped to the viral genome in which a given nucleotide was present. The x-axis indicates the nucleotide position on the viral genome. A colinear schematic HIV-1 genome is presented above each set of charts. (C). Expanded view of read densities for the 400 nucleotides at the 5' end of the viral genome. For (B) and (C), CLIP assays for each construct were repeated in at least two independent experiments, and the average read density is plotted.

Fig.S1
that the dimerized RNA elements are part of the structure recognized by Gag. Overall, these results validate the utility of the CLIP method used herein, and are consistent with the finding that the tertiary, and to some extent the quaternary, structure of the Ψ element is important for accurate recognition by Gag.

HIV-1 capsid is required for specific binding to Ψ
We next compared the ability of various manipulated CANC proteins to bind to the gRNA in CLIP assays ( that NC is required for the specific interaction between Gag and Ψ (Aldovini and Young, 1990;Didierlaurent et al., 2011;Dorfman et al., 1993;Gorelick et al., 1990).
Next, to test whether an isolated NC domain was sufficient for specific binding to Ψ, we deleted the region encoding the CA domain from the CANC construct, thereby generating a construct that encodes NC (along with the SP2-p6 C-terminal extension) with an N-terminal 3xHA tag. CLIP experiments using this construct revealed that NC was not sufficient to bind Shaded regions indicate NC zinc fingers (B) Read density distribution on viral RNA from CLIP experiments in which constructs encoding CANC, and mutant derivatives with deletions of the zinc finger 1 (CANC dZF1), zinc finger 2 (CANC dZF2), or both zinc fingers (CANC dZF) were used. Alternatively, a construct in which CA was deleted (NC) was used. Each chart represents at least two independent experiments, and the average read density is plotted.
(C) Western blot analysis of Gag-derived proteins following chemical crosslinking in living cells using bismaleimidohexane (BMH) prior to cell lysis. Proteins were detected with an anti-NC antibody.
(D) Read density distribution on viral RNA from CLIP experiments in which constructs encoding chimeric NC proteins with artificial multimerizing domains were used. Each chart represents at least two independent experiments, and the average read density is plotted.  specifically to Ψ. Instead, low read counts, distributed across the entire HIV-1 genome, were obtained ( Figure 2B). Indeed, the deficit in specific Ψ binding associated with CA deletion was as profound as the deficit associated with deletion of both NC zinc fingers ( Figure 2B).
Since CANC may form multimers, we considered the possibility that multimerized NC may be sufficient to enable Ψ-specific RNA binding. Thus, to test whether CA-induced multimerization could be recapitulated by heterologous protein-protein interaction domains that can drive hexamerization, we substituted the CA domain in the CANC constructs. The

CA driven assembly of a nascent immature Gag lattice is required for specific Ψ recognition
To investigate how CA drives the specific interaction between CANC and Ψ, we designed twenty-one CA substitution mutants. These substitution mutants were selected based on the structures of HIV-1 immature Gag lattice and mutagenesis studies of residues important for mature or immature CA assembly (Forshey et al., 2002;Ganser-Pornillos et al., 2004;Schur et al., 2016;von Schwedler et al., 2003von Schwedler et al., , 1998Wagner et al., 2016).   These substitutions targeted interfaces at either the 2-fold, 3-fold or 6-fold axes of symmetry, where interactions required for the formation of the complete immature Gag lattice occur (Figure 3 B-E). Each mutant was subjected to three types of experiment: First, in vivo chemical crosslinking was performed to test propensity of mutant CANC proteins to multimerize. Second, virus particle production experiments were done in which the CA substitutions were introduced to HIV-1-NL4-3 wild-type construct and Gag processing/extracellular virion formation tested. Finally, the panel of CANC mutants was subjected to CLIP experiments to test for specific binding to Ψ.
To test whether the CA mutants could support lattice assembly that would lead to the generation of extracellular virions, we introduced the same set of CA substitutions into a fulllength infectious HIV-1 NL4-3 proviral construct and assessed virion production by proviral plasmid transfected 293T cells. This analysis revealed that mutants such as R18A/N21A, P38A, E45A, Q219A, SP1 T8I, and SP1 T12A, generated virions at, or close to, wild-type levels, while other mutants either generated reduced levels of virions or failed to generate virions ( Figure 4-figure supplement 1). These results are consistent with previous studies (von Schwedler et al., 2003). Results from the in vivo CANC chemical crosslinking experiment and virion production experiments showed a correlation between the ability of a given CA mutant to affect CANC multimerization and virion production: mutants that formed high order CANC multimers produced more virions than mutants in which CANC multimerization was impaired When CANC proteins encoding the same panel of mutants were subjected to CLIP experiments, the mutants could be divided into two categories. One group of mutants bound to Ψ with high specificity; this group included R18A/N21A, P38A, E45A, Q219A, SP1 T8I, and SP1 T12A. A second group included all mutants other than the aforementioned group of six,

. Effect of CA mutations on CANC RNA binding specificity
Read density distribution on viral RNA from CLIP experiments in which constructs encoding CANC mutants were used. Each chart represents at least two independent experiments, and the average read density is plotted. correlation between CA multimerization and Ψ-specific binding: mutants that were readily able to form high order multimers bound to Ψ with high specificity, similar to the wild-type CANC (in the cases of R18A/N21A, P38A, E45A, Q219A, SP1 T8I, and SP1 T12A).
Conversely, mutants that had deficits in multimerization failed to bind specifically to Ψ.
Notably, impairment of Ψ binding imposed by CA substitutions occurred irrespective of which interface (2-fold, 3-fold, or 6-fold) was targeted. Overall, these experiments suggest that the ability of CANC to form a nascent immature lattice is important for specific recognition of Ψ.

Deficits in Ψ recognition are exhibited by inositol hexakisphosphate binding-deficient mutants but are restored in second-site revertants
The abundant intracellular small molecule inositol hexakisphosphate (IP6) coordinates two rings of positively charged lysine residues formed by K158 and K227 at the base of the CA hexamer (Dick et al., 2018). As such, it is required for the assembly of the immature Gag lattice in cells. To corroborate the above findings that the formation of a nascent immature lattice is required for specific Ψ-binding, we performed CLIP experiments using two mutants (CANC K158A and CANC K227A) that are impaired for IP6 coordination and are thus incapable of particle assembly. Both CANC K158A and CANC K227A mutants exhibited deficits in Ψ-binding ( Figure 5A). A second-site revertant of K158A and K227A, specifically a lattice stabilizing substitution in SP1(SP1 T8I), restores virion assembly and viral infectivity (Mallery et al., 2021;Poston et al., 2021). Notably, introduction of this second site substitution (in CANC K158A/SP1 T8I and CANC K227A/SP1 T8I) enabled otherwise Ψ-biding defective CANC proteins to bind Ψ with high specificity (Figure 5B and 5C). Thus, these results reinforce the conclusion that the specific binding to Ψ requires the correct assembly a the nascent viral immature capsid lattice.

Discussion
Overall, we conclude that the initiation of the assembly of the immature HIV-1 Gag lattice in the cytosol of infected cells is required for the specific initial interaction between Gag/CANC and the Ψ element of the gRNA. Nevertheless, questions remain about the precise number of Gag/CANC monomers required to bind to Ψ specifically. Imaging studies of the HIV-1 virion assembly process suggest that a small number of Gag molecules (below the limit of detection by fluorescent microscopy) are involved in initial Gag/gRNA complex formation (Hendrix et al., 2015;Jouvenet et al., 2009;Kutluay and Bieniasz, 2010). However, the ability of CANC to multimerize into higher-order multimers (such as 10-mers or greater) in our in vivo chemical crosslinking experiment, predicted specific Ψ binding, suggesting that the 2-fold, 3-fold, and 6-fold interactions interfaces are all required, and that perhaps high-order multimers of CANC may be needed to recognize Ψ (see below). Several lines of evidence from previously published work are consistent with this proposition: 1) SHAPE and XL-SHAPE experiments identified at least ten potential Gag/NC interaction sites in gRNA which are important for packaging (Kenyon et al., 2015;Wilkinson et al., 2008); 2) Electrophoretic mobility shift experiments suggest that at least 20 Gag molecules were needed to form a complete Gag/leader gRNA dimer complex (D'Souza et al., 2021); 3) Studies of NC and Ψ interactions with NMR and ITC suggests that there are at least two dozen high-affinity NC binding sites in a dimerization competent form gRNA (Ding et al., 2020). Further studies are thus needed to determine the precise stoichiometry between Gag and Ψ interaction at the initiation of Ψ binding and particle assembly.
There were some unexpected findings from our chemical crosslinking experiments. For example, the W184A/M185A mutant was mainly monomeric under the crosslinking conditions in our assay, despite the fact that W184/M185 are located at an inter-hexamer interface rather than an intra-hexamer interface. In a previous report, a CA quadruple mutant A14C/E45C/W184A/M185A was able to form a hexamer under reducing conditions (Pornillos et al., 2009). Possible explanations for this discrepancy include (i) the A14C/E45C/W184A/M185A CA mutant was purified and crosslinked in vitro, while in our study, W184A/M185A crosslinking was attempted in cells; (ii) the A14C/E45C/W184A/M185A CA mutant assembled through mature lattice interactions, whereas herein the W184A/M185A mutant was constrained by N-and C-terminal extensions to form the immature lattice, which involves different interfaces. Another unexpected finding from our in vivo chemical crosslinking studies was that certain mutants, such as A22D and A42D, were mainly monomeric under in vivo crosslinking conditions (Schur et al., 2016;Wagner et al., 2016).
A22D and A42D were expected to form dimers or hexamers because these substitutions disrupt the three-fold CA-CA interaction interface rather than the two-fold or the six-fold CA-CA interaction interfaces. This result, along with the fact that the dimer interface mutants E28A/E29A and W184A/M185A mutants were also mainly monomeric in cells, suggests that the basic building block of HIV-1 immature lattice formation is a Gag dimer rather than a Gag hexamer: mutants that failed to form a dimer via inter-hexamer contacts also failed to form a hexamer. This suggestion is partially consistent with models (Grime and Voth, 2012;Tomasini et al., 2018;Tsiang et al., 2012) which propose that a trimer-of-dimers of CA is the basic building block of the HIV-1 immature lattice. However, crosslinking results for A22D and A42D mutants that target the trimer interface are not consistent with the trimer-of-dimers model, because these two mutants do not to form crosslinkable dimers, despite the fact that these substitutions are not at the two-fold interface. Overall, the results of the crosslinking experiments suggest that all the three CA-CA interaction interfaces (two, three, and six-fold interaction interfaces) contribute simultaneously to immature lattice formation, since disruptions of either two-fold (W184A/M185A), or three-fold (A22D and A42D), or six-fold (D197A, G222A, P224A, and SP1 M4A) CA-CA interaction interfaces lead to overall defects rather than the formation of discrete low order multimeric species.
Overall, our study shows that CA is essential for the specific interaction between HIV-1 Gag and Ψ. CA is therefore key, not only for controlling the morphology of HIV-1 particle assembly but also for selective viral genome packaging. Based on the evidence that HIV-1, HIV-2, and SIV Gag can co-assemble and package each other's genome (Al Shamsi et al., 2011;Franke et al., 1994;Motomura et al., 2008;Rizvi and Panganiban, 1993) and the conservation of primary and tertiary Gag structures between these viruses, the insights gained from this study are likely applicable across primate lentiviruses. Whether the findings described herein constitute a more generalized principle, and that formation of nascent capsid or nucleocapsid lattices enables specific interactions between virion proteins and genomes in the assembly of other RNA viruses, remains to be determined.

Plasmids and Cells
Constructs for CLIP experiments were generated based on a previously described HIV proviral construct, NL4-3 (MA-3xHA/PR-), which contains a 3xHA tag within the stalk region of MA (between residues 127 and 128) and an inactivating mutation (D81A) in the viral protease (Kutluay et al., 2014). The CANC constructs were generated by changing the start codon (ATG) of the MA domain of NL4-3 (MA-3xHA/PR-) to ACG to abrogate the expression of MA, thus translation begins at an AUG at the amino terminus of the 3xHA tag. CANC variants were generated by overlapping PCR to introduce substitutions in the CA domain.
The NC construct was generated by overlapping PCR to delete the CA domain in the CANC construct. GCN4pII, GCN4pAA, and ccHex2 chimeric constructs were generated by overlapping PCR to replace the CA domain in the CANC construct (but retaining the CA Cterminal residues "223GPGHKARVL231" intact for CASP1 helix formation) with GCN4pII or GCN4pAA leucine zippers, or with ccHex2 synthetic peptide. For GCN4pII-SP1-NC constructs, the amino-acid sequence and junctions are as follows: RMKQIEDKIEEILSKIYHIENEIARIKKLIGERTSGPGHKARVL, where the GCN4pII is in bold, the CA C-terminal residues in italic, and a two amino acids "TS" linker in between. For GCN4pAA-SP1-NC constructs, the amino-acid sequence and junctions are as follows:

MKVKQLADAVEELASANYHLANAVARLAKAVGERGSGPGHKARVL,
where the GCN4pAA is in bold, the CA C-terminal residues in italic, and a two amino acids "GS" linker in between. For ccHex2-SP1-NC constructs, the amino-acid sequence and junctions are as follows: GEIAKSLKEIAKSLKEIAWSLKEIAKSLKGSGPGHKARVL, where the ccHex2 is in bold, the CA C-terminal residues in italic, and a single amino acid "S" in between. In the virion production assay, substitution mutations were introduced into the capsid domain of the HIV-1 NL4-3 wild-type proviral plasmid. HEK293T cells were transfected with these constructs for CLIP experiments, in-cell chemical crosslinking experiments, and virion production experiments.

CLIP-seq
HEK293T cells at 90% confluency in a 15-cm dish were transfected with 25 ug proviral plasmids using polyethylenimine (PEI). Cell culture media was replaced with fresh media 10-14 hours after transfection. Ribonucleoside analog 4-thiouridine (4SU) was added to the media 12-14 hours before UV crosslinking at a final concentration of 100 μM. On the day of crosslinking, cells were rinsed with Phosphate-Buffered Saline (PBS) once and cross-linked with 500mJ/cm 2 UV (λ = 365 nm) in a Boekel UV crosslinker. After UV crosslinking, cells were resuspended in 15 ml PBS and cell pellets were collected at 500 x g for 5 minutes of centrifugation. Cell pellets were stored at -80℃ until use. The CLIP procedure was modified based on a previously published protocol (Kutluay et al., 2014;Shema Mugisha et al., 2020) with modifications. Modifications are in two areas: (i) a 3' adapter containing an infrared dye (Zarnegar et al., 2016) was used instead of a 3' adapter labeled with 32 P; (ii) the 3' adapter was ligated to protein/RNA complexes on antibody-conjugated Dynabeads (ThermoFisher 10004D) instead of ligating to purified RNAs. Rnase A/T1 mix (ThermoFisher EN0551) was used at a dilution of 1:100 to digest RNAs in the cell lysate before immunoprecipitation. For immunoprecipitation CLIP constructs, we used anti-HA mouse IgG (BioLegend, 901503).
Alternatively, in experiments with chimeric NC fusion proteins, we used a custom rabbit polyclonal IgG antibody raised against the NC domain of the HIV-1 NL4-3.

Bioinformatic analysis
The CLIP library was sequenced with Illumina Nextseq 500 platform. Raw fastq reads were processed with Cutadapt (Martin, 2011), and reads that were fewer than 20 nt, did not contain the 3' adapter, or contained ambiguous nucleotides were excluded. Barcodes were collapsed and trimmed with the FASTX toolkit (http://hannonlab.cshl.edu/fastx_toolkit) prior to mapping.
Reads were mapped using Bowtie (Langmead et al., 2009) to the corresponding viral genomes in CLIP experiments. SAMtools (Li et al., 2009) and in-house scripts (Kutluay et al., 2014) were used to generate counts of each base in the viral genomes. Read density was calculated from the counts of each base divided by the total counts of bases mapped to the corresponding viral genome. Read density graphs were generated with GraphPad Prism 9 (www.graphpad.com). The Ψ binding specificity was quantified by dividing the number of reads mapped in the Ψ region by the total number of reads mapped to the viral genome.

In vivo bismaleimidohexane crosslinking
The use of cysteine-specific crosslinker bismaleimidohexane (BMH) to study CANC interactions in cells was based on earlier studies of CANC assembly in vitro (Hansen and Barklis, 1995;McDermott et al., 1996). In our study, the BMH crosslinking method was modified to study CANC interactions in cells. Specifically, 1 x10 6 HEK293T cells were seeded in each well of a six-well plate one day prior to transfection. Then 2 μg of pCANC and variants thereof were transfected with PEI and culture media was replaced with fresh media 7-10 hours after transfection. Cells were washed once with PBS 20-24 hours post-transfection and suspended with 1ml PBS/EDTA (5 mM) (cell density was around 2.5-3 x10 6 /ml). Next, 200 μl cell suspension was used for bismaleimidohexane (BMH) crosslinking whereby 5 μl of freshly dissolved BMH (40 mM stock in DMSO, ThermoFisher 22330) was added to 200 ul cell suspension (final BMH concentration 1 mM) to start the crosslinking reaction. DMSO mock-treated reactions were simultaneously performed. The reactions were incubated in the dark at room temperature for 1 hour. Then, 7 μl 1M DTT was added to the reactions for 15 minutes of incubation to stop the reactions. Next, 72 μl 4xNuPAGE™ LDS Sample Buffer (ThermoFisher NP0008) was added to the reaction followed by 15 min incubation. The samples were sonicated and heated at 72℃ for 10 minutes before loading 20 μl/well on NuPAGE™ 3 to 8% Tris-Acetate gels (ThermoFisher, EA03785BOX) alongside high molecular weight markers (ThermoFisher LC5699). The gel was run at 150 Volts for 60 minutes. Gels were transferred in the transfer buffer (ThermoFisher NP0006) with 20% were imaged at the Licor Odyssey imaging system.

Virion production assay
HEK293T cells (3 x10 5 ) were seeded in each well of a twenty-four-well plate. Proviral plasmids (1ug HIV-1 NL4-3 wild type or CA mutant) were transfected with PEI the next day. Cultural media was replaced with fresh media one day after transfection. At 40-48 hours after transfection, 600 ul virion supernatant from each sample was filtered with a 0.2 μm filter and gently placed above an equal volume of 20% sucrose/PBS cushion in a 1.5 ml Eppendorf tube. Virions were pelleted by centrifugation at 14000rpm for 1.5 hours at 4 ℃. Supernatant was removed and 50 μl 1x NuPAGE™ LDS Sample Buffer was added to lyse pelleted virions. Virion samples were heated 72℃ for 10 minutes and 15 μl virion lysate was loaded into each well of a NuPAGE™ 4 to 12% Bis-Tris gels (ThermoFisher NP0329BOX). For cell lysates, 200 ul 1x NuPAGE™ LDS Sample Buffer (ThermoFisher NP0008) were added to each well, sonicated and heated at 72℃ for 10 minutes before loading into the NuPAGE™ 4 to 12% Bis-Tris gels. Blotted membranes were probed with mouse monoclonal anti-HIV-1 p24CA (183-H12-5C, NIH AIDS Reagent Program) and rabbit HSP90 Polyclonal antibody (Proteintech 13171-1-AP).

Acknowledgments
We thank the staff of the Rockefeller University Genomics Resource Center for assistance