Virus specificity and nucleoporin requirements for MX2 activity are affected by GTPase function and capsid-CypA interactions

Human myxovirus resistance 2 (MX2/MXB) is an interferon-induced GTPase that inhibits human immunodeficiency virus-1 (HIV-1) infection by preventing nuclear import of the viral preintegration complex. The HIV-1 capsid (CA) is the major viral determinant for sensitivity to MX2, and complex interactions between MX2, CA, nucleoporins (Nups), cyclophilin A (CypA), and other cellular proteins influence the outcome of viral infection. To explore the interactions between MX2, the viral CA, and CypA, we utilized a CRISPR-Cas9/AAV approach to generate CypA knock-out cell lines as well as cells that express CypA from its endogenous locus, but with specific point mutations that would abrogate CA binding but should not affect enzymatic activity or cellular function. We found that infection of CypA knock-out and point mutant cell lines with wild-type HIV-1 and CA mutants recapitulated the phenotypes observed upon cyclosporine A (CsA) addition, indicating that effects of CsA treatment are the direct result of blocking CA-CypA interactions and are therefore independent from potential interactions between CypA and MX2 or other cellular proteins. Notably, abrogation of GTP hydrolysis by MX2 conferred enhanced antiviral activity when CA-CypA interactions were abolished, and this effect was not mediated by the CA-binding residues in the GTPase domain, or by phosphorylation of MX2 at position T151. We additionally found that elimination of GTPase activity also altered the Nup requirements for MX2 activity. Our data demonstrate that the antiviral activity of MX2 is affected by CypA-CA interactions in a virus-specific and GTPase activity-dependent manner. These findings further highlight the importance of the GTPase domain of MX2 in regulation of substrate specificity and interaction with nucleocytoplasmic trafficking pathways.


Introduction
Innate immune detection of viral infection results in the production of type I interferons (IFNs), which in turn induce the expression of hundreds of IFN-stimulated genes (ISGs) that confer an 'antiviral' state, impeding the propagation of many viruses (1,2).Among the antiviral ISGs are the dynamin-like guanosine triphosphatases (GTPases) MX1 (MXA) and MX2 (MXB).Both proteins contain an amino-terminal GTPase domain connected to a carboxy-terminal stalk domain via a tripartite bundle signaling element (BSE) (3).MX1 is exclusively found in the cytoplasm, while MX2 contains a nuclear localization signal (NLS)-like sequence in its first 25 amino acids and localizes at the nuclear pore complex (NPC) (4)(5)(6).MX1 has long been known as a broadly acting and potent antiviral protein (3), while MX2 was ascribed with antiviral function in 2013 as an inhibitor of HIV-1 and other primate lentiviruses, and later shown to also restrict replication of herpesviruses and hepatitis B and C viruses (6)(7)(8)(9)(10)(11).
MX2 inhibits HIV-1 infection prior to the chromosomal integration of proviral DNA, but after the completion of reverse transcription (6,7,12), and current models suggest that it acts by preventing nuclear import of the viral preintegration complex.The viral capsid (CA) is the major viral determinant of MX2 sensitivity, and several single amino-acid substitutions in CA have been identified that confer partial or complete resistance to MX2 (6-8, 13, 14).MX2 has also been found to directly bind the HIV-1 CA, however the relevance of this binding for viral inhibition is unclear since MX2-resistant CA proteins are still efficiently bound by MX2 (15,16).
The antiviral activity of MX1 has been extensively investigated, and current understanding indicates that engagement of MX1 with components of viral replication complexes (such as the nucleoprotein of orthomyxoviruses) results in higher order oligomerization, followed by GTP hydrolysis and conformational changes which lead to the perturbation of viral transcription/replication via mislocalization of targeted components, blocking nuclear translocation, and/or disruption of the functional integrity of the replication complex.GTPase function and higher order oligomerization are generally required for antiviral activity of MX1 (3).
Conversely, GTP binding and hydrolysis are dispensable for the antiviral activity of MX2 (6,7,17), and although dimerization is essential, higher order oligomerization is not required for viral restriction (16,18).Finally, while anti-viral specificity of MX1 is largely determined by a disordered loop (L4) in the stalk domain (19), antiviral specificity of MX2 is determined by the N-terminal domain (NTD) (13,17), indicating that the mechanisms underlying inhibition of viral infection by MX1 and MX2 are distinct.The antiviral activity of MX2 is also regulated by protein phosphorylation.Phosphorylation of serines 14, 17, and 18 in the NTD suppresses antiviral activity and alters interactions with the viral CA and accumulation at the nuclear envelope, while dephosphorylation by myosin light chain phosphatase (MLCP) upon IFN stimulation restores antiviral function (20).Phosphorylation status of additional residues throughout all domains of MX2 also alter the antiviral activity and specificity of MX2 (21).Thus, a variety of determinants throughout MX2 modulate its function and viral specificity.
Sensitivity of HIV-1 to MX2 activity is affected by complex interactions between CA and cellular proteins involved in the early stages of HIV-1 infection, including nucleoporins (Nups) and the peptidyl-prolyl isomerase cyclophilin A (CypA) (reviewed in (22)).CypA-CA interactions can affect many early steps of HIV-1 replication, disruption of the interaction between HIV-1 and CypA, either genetically or by cyclosporine A (CsA) addition, reduces the efficiency of HIV-1 infection (reviewed in (23)), and some studies have indicated that CypA-CA interactions influence nuclear entry (24,25), and more recently CypA has been shown to protect the HIV-1 CA from restriction by TRIM5⍺ (26,27).However, it remains unclear whether the isomerase activity of CypA plays a role in its pro-viral function, since mutations that abolish enzymatic activity also affect CA-binding.Indeed, defining a precise role for CypA in HIV-1 infection has proved difficult because CsA treatment has diverse enhancing and inhibiting effects on infection that are determined by CA mutations and target cell type, suggesting that the function of CypA in HIV-1 infection is differentially affected by other cellular proteins in various contexts (reviewed in (23,28)).
Here, we investigated the effects of CA-CypA interactions on the antiviral activity and specificity of MX2.We find that the effects of CypA on MX2 activity against HIV-1 are the direct result of CA-CypA interactions.We also identified cell-type-dependent determinants for the GTPase and oligomerization requirements for MX2 activity.We show that abrogation of GTP hydrolysis by MX2 conferred enhanced antiviral activity when CA-CypA interactions were abolished.This activity is independent of previously reported CA-binding residues in the GTPasedomain of MX2.Finally, we show that GTPase-deficient MX2 has altered nucleoporin requirements for antiviral activity.These results indicate that GTPase function alters substrate specificity and antiviral function of MX2.
Therefore, to address whether there are capsid-independent effects of CypA on HIV-1 infection or MX2 activity, we generated CypA knock-out cell lines as well as cells expressing CypA from its endogenous locus but with specific point mutations introduced to alter CA-binding specificity.
TRIMCyp from both rhesus and pigtailed macaques does not restrict HIV-1 infection and differ from human CypA at only two amino acid positions [D66N and R69H], both of which are outside the active site (Fig 1A) and differently affect binding to lentiviral capsids (34,35).We first generated cells expressing HA-tagged owl monkey TRIMCyp (omkTRIMCyp), or chimeric proteins in which the CypA domain was replaced with human CypA (TRIM-huCypA) or human CypA with D66N, R69H, or both D66N/R69H substitutions.We then challenged these cells with HIV-1WT, its derivatives encoding CA mutations with altered MX2-sensitivity, and other lentiviruses (S1 Fig) .Cells were also treated with the CypA inhibitor cyclosporine A (CsA) at the time of infection to abolish CypA:CA interactions.As expected, HIV-1WT infection was potently inhibited (~200-fold) by TRIM-huCypAD66N and TRIM-huCypAD66N/R69H in a manner reversible by CsA treatment, while TRIM-huCypAR69H was inactive against HIV-1WT.With the exception of the cyclophilin-binding loop mutant G89V, all HIV-1 CA mutants tested exhibited similar sensitivity to the chimeric TRIM-huCypAs as the WT virus.HIV-2 on the other hand, was inhibited (~10-fold) by TRIM-huCypAD66N and TRIM-huCypAD66N/R69H, but not TRIM-huCypAR69H, while SIVmac was insensitive to all TRIMCyps since it does not bind cyclophilins (S1 Fig) (25,36,37).CypA mutant cell lines were then transduced with a lentiviral vector for doxycyclineinducible expression of MX2 and infected with GFP-reporter viruses in the presence or absence of CsA (Fig 1).CsA addition in HT1080 cells is known to reduce infection of HIV-1WT and abolish the antiviral activity of MX2 ((29) and Fig 1B).We observed similarly reduced levels of infectivity and a lack of MX2 sensitivity in CypA -/-, CypAD66N, and CypAD66N/R69H, but not CypAR69H cells.
Mutation or deletion of CypA had no effect on infection of the HIV-1G89V CA, or on the slight enhancement of infection by this mutant upon MX2 expression.We previously reported a dramatic sensitivity of the cell-cycle-dependent and MX2-resistant HIV-1N57S CA to CsA addition that is reversed by MX2 (29).Here we observed the same ~50-fold reduction in infectivity and rescue by MX2 in CsA-treated and CypA -/-, CypAD66N, or CypAD66N/R69H, but not CypAR69H cells (Fig 1B).Since the N57A CA mutation was utilized for in vitro CA binding experiments, we also confirmed that this mutant exhibited the same phenotype, as did the N57D mutation, although this mutant had lower overall levels of infectivity which were slightly enhanced by MX2 even in the absence of these data demonstrate that the effect of CsA on HIV-1 infection and MX2 sensitivity are the direct result of abrogating CA-CypA interactions and do not involve other cyclophilins, and further suggests that they do not involve other cellular functions of CypA.However, since it is unknown whether the conformational changes in CypA resulting from substitution at residue 66 affect interactions between CypA and any of its cellular substrates, we cannot definitively exclude the possibility that CypAD66N fails to isomerize a cellular substrate relevant for these interactions.
In contrast to HIV-1, HIV-2 sensitivity to MX2 is unaffected by CsA treatment ((29) and Fig 1B), although HIV-2 CA does interact with CypA with low affinity (34).The CypA D66N and R69H mutations increase binding affinity, making HIV-2 sensitive to rhesus and pigtailed macaque TRIMCyps ( (34,35,37) and S1 Fig) .On the other hand, huCypA interaction with SIVagmTAN do not appear to involve CypA residues 66 or 69, as this virus was restricted by all TRIMCyp fusions tested (although this virus is not restricted by owl monkey TRIMCyp with the NH mutation, highlighting the complexity of lentiviral CA-CypA interactions (35)).However, both HIV-2 and SIVagmTAN infection and MX2 sensitivity were unaffected in CypA-knockout and mutant cells, and limited oligomer forming R455D mutants exhibited no anti-HIV-1 activity, while dimeric YRGK487-490AAAA had extremely reduced antiviral activity in both HeLa and HT1080 cells.This is in contrast with previous reports that have shown full antiviral activity of dimeric MX2 in U87-MG and HOS cells (16,18) and suggests that the requirements for MX2 oligomerization are cell-type specific.The T151A mutant of MX2, which binds, but does not hydrolyze GTP (5), restricted HIV-1 infection in both HeLa and HT1080 cells, confirming that GTPase activity is dispensable for anti-HIV-1 activity (6, 7).The T151A mutant also restricted HIV-1WT infection in the presence of CsA in both cell lines.On the other hand, the GTP-binding mutant K131A, which retains anti-HIV-1 activity in K562, HOS, U87-MG (6, 7), and HeLa cells (S5 Fig), failed to inhibit infection in HT1080 cells, indicating that requirements for GTP binding are also cell-type specific.The requirement for GTP binding is also virus-specific, since the K131A mutation abrogated antiviral activity against HIV-2 and SIVmac, while all other MX2 mutants that inhibited HIV-1WT infection also inhibited HIV-2 and SIVmac infection.
In most cases, MX2 mutants that inhibited HIV-1WT infection also enhanced HIV-1G89V CA infection in both HeLa and HT1080 cells, while those that did not inhibit the WT virus had no effect on the G89V CA mutant virus (Fig 3 and S5 Fig).Similarly, only MX2 mutants that inhibited HIV-1WT infection (MX1MX2-NTD and SSS14,17,18AAA) were able to reverse the reduction in HIV-1N57S CA infectivity in the presence of CsA in HT1080 cells.These data suggest that similar determinants are involved in the antiviral activity of MX2, as well as the CA-specific, CypA-dependent pro-viral effects of MX2.The MX2T151A mutant, however, was a notable exception to this trend in that it failed to rescue HIV-1N57S CA infection in CsA-treated HT1080 cells, and inhibited HIV-1G89V CA infection in both HeLa and HT1080 cells.Thus, while CA-CypA interactions are required for the full antiviral activity of WT MX2, MX2 which can bind but not hydrolyze GTP restricts HIV-1 infection even in the absence CypA.
We next tested the effects of GTPase-deficient MX2 on HIV infection in our CypA knockout and mutant cell lines.As expected, the MX2T151A mutant inhibited both HIV-1WT and HIV-1G89V CA infection in the presence or absence of CsA in control, CypA -/-, and all CypA mutant cell lines CA.Thus, these data indicate that the antiviral activity MX2T151A has distinct requirements for Nups and NTRs, some of which are also affected by CA-CypA interactions.

Discussion
While the NTD of MX2 is of principal importance for viral substrate recognition and interaction with cellular proteins (6, 9-13, 15, 17, 38), there is a growing appreciation for the importance of the GTPase domain in MX2 function (21,39,40).Here, we demonstrate that the GTPase activity of MX2 affects substrate specificity and nucleoporin requirements for anti-HIV-1 activity.While WT MX2 does not restrict HIV-1 in the absence of CypA binding to CA, the T151A MX2 mutant, which is able to bind, but not hydrolyze GTP ( 5 therefore there are additional determinants unique to MX2 that affect phosphorylation, oligomerization/conformation, CA recognition, or interaction with other cellular proteins which alter antiviral activity and substrate specificity.Importantly, we found that nucleoporin requirements of the T151A MX2 mutant for antiviral activity were altered as compared to WT MX2 in both CypAdependent and -independent ways, suggesting that this mutation affects both antiviral substratespecificity, and interaction with nuclear pore complexes.Additionally, since MX2 also affects nuclear import of some non-viral cargos, it will be of interest in future investigations to determine whether the determinants identified here which affect viral specificity also alter inhibition of specific nuclear import pathways.Therefore, it is likely that recognition of HIV-1 and other primate lentiviruses by MX2 involve distinct interactions with the capsid surface, consequently requiring distinct determinants.Indeed, given the ability of MX2 to inhibit replication of highly divergent viruses, including herpesviruses and flaviviruses (reviewed in (22)), it appears that we are just beginning to understand the mechanisms regulating MX2 specify and activity.
We also identified here a number of MX2 determinants that are cell-type specific, highlighting the importance of other cellular players in MX2 activity.While our previous investigation found that the K131A mutant of MX2 which is unable to bind GTP (5), retains antiviral activity (6, 7), here we found that this activity is cell-type dependent since this mutant is inactive in HT1080 cells (Fig 3).We further find that the YRGK487-490AAAA mutant of MX2, has minimal antiviral activity in either HT1080 or HeLa cells, demonstrating that the requirements for higherorder oligomerization of MX2 are also cell-type dependent.While the MX2 stalk is the major driver of oligomerization, the GTPase domain also exerts effects on MX2 conformation (40).
Biochemical studies have found that MX2 spontaneously forms helical assemblies that are depolymerized into oligomers upon GTP binding; importantly, the T151A MX2 mutant also selfassembles, however these assemblies are loosened, but not dissociated upon GTP binding (40).
Thus, conformational changes in MX2 may explain the expanded substrate-specificity of the T151A mutant.Importantly, recent work has shown that the MX2 residue T151 is phosphorylated, and that the phosphomimetic T151D mutant has increased antiviral activity (21).We recapitulated this finding and additionally found that this mutant is also able to restrict HIV-1 infection in the mutant is unlikely to bind GTP, as noted by Betancor, G. et al, the negative charge of the phosphomimetic T151D mutant may resemble the GTP-bound T151A mutant, and both mutants have distinct subcellular localization as compared to WT MX2 (21), they may both therefore exist in a "loose helical assembly".While high GTP concentrations within cells would suggest that MX2 exists in its GTP-bound non-assembled state (40), it is also possible that changes in the phosphorylation state of T151 regulates MX2 conformation (and GTP binding), thereby affecting interactions with both viral substrates and nuclear pore complexes.
In summary, this report highlights the importance of GTPase activity in modulation of the substrate specificity of MX2 and further indicate that highly complex interactions between MX2 and its cellular environment determine antiviral activity.While we have focused specifically on human MX2, the regulation of the antiviral activity of other MX proteins is similarly complex.Given the importance of MX proteins in the innate immune responses of a highly divergent range of vertebrate species (3), the evolution of Mx genes has likely been the result of complex selective pressures balancing substrate breadth and specificity, as well as interaction with cellular partners.

TRIM5-fusions
An LHCX MLV vector (Clontech) was engineered to express the N-terminal domains (RIGN, B-Box and coiled coil) from owl monkey (omk) TRIMCyp followed by an HA tag and cloning sites (NotI/SalI) that allowed introduction of various proteins as described in (41,42)

HIV-1 CA mutants
HIV-1 GFP reporter viruses containing the N57A or N57D capsid mutation (HIV-1N57A and HIV-1N57D) was generated by overlap PCR (primers listed in S1 Table ) and insertion into pNHGCapNM as described previously (31) using NotI and MluI sites.

Cell lines
HEK 293T, HeLa, and HT1080 cell lines were maintained in Dulbecco's Modified Eagles Medium [(DMEM) Gibco] with 10% fetal calf serum (Gibco), gentamicin (Gibco), and Plasmocin® prophlyactic (InvivoGen).Plasmocin® was not present in cultures during infection, transduction, or transfection.Cells were purchased from ATCC and were assumed to be authenticated by their supplier and were not further characterized.Cells were monitored quarterly for retroviral contamination by SYBR-Green based PCR RT assay (43,44) and tested for mycoplasma contamination by MycoStrip® detection kit (InvivoGen).Derivatives of HeLa and HT1080 cells containing doxycycline-inducible Mx2, or fusion proteins were generated by transduction with LKO-derived lentiviral vectors (13) followed by selection in 1μg ml -1 puromycin (Sigma-Aldrich).
HeLa cells stably expressing TRIM fusions were generated by transduction with LHCX-derived vectors followed by selection in 100μg ml -1 hygromycin B (Gibco).Vector stocks for transduction were generated by co-transfection of 293T cells with a VSV-G expression plasmid, an HIV-1NL4-3 Gag-Pol expression plasmid, and LKO-derived vector, or an MLV Gag-Pol expression plasmid and an LHCX-derived vector using polyethyleneimine (PolySciences).Expression was induced in pLKO transduced cell lines through an overnight treatment with 500ng/ml doxycycline hyclate (Sigma-Aldrich) prior to challenge with retroviruses or retroviral vectors.
Custom Alt-R TM CRISPR Cas9 guide RNAs targeting the PPIA locus were designed using the IDT website.RNPs containing crRNA 1, 2, or 3:tracerRNA duplexes were generated according to manufacturer instructions: 1μL of 100 μM crRNA, 1μ of 100μM tracrRNA-ATTO TM 550 (IDT) in 98μL of Nuclease-Free Duplex Buffer (IDT) were heated at 98 o C for 5min, then cooled to room temperature.15μL of annealed guide RNA oligos were mixed with 15μL of 1μM recombinant Cas9 (IDT) in 220μL of Opti-MEM media (Gibco) and incubated for 5min at room temperature, complexes were used immediately or stored at -80 o C. HT1080 cells were reverse transfected in 96-well plates with RNPs using RNAiMax (Thermo Fisher) according to manufacturer instructions, transfection complexes containing 15μL of each RNP was mixed with 1.2μL of RNAiMax and 18.8μL of OptiMEM for 20mins at room temperature, followed by addition of 4x10 4 cells/well.For generation of point mutant cell lines, 1μM Alt-R TM HDR Enhancer V2 (IDT) was included.HT1080 cells were transfected with RNPs containing guides 1 and 3 to generate CypA -/-cells and guides 2 and 3 to generate CypA-mutant cells (S2 Fig) .Control cells were transfected with RNPs containing the IDT negative control crRNA.16hours post-transfection, cells were transferred to a 24-well plate, and AAV infection/transduction was repeated after an additional 24-48hours.
Single-cell clones were then derived by limiting dilution.
CypA -/-clones were screened via western blotting, sequences of knock-out alleles were determined following PCR amplification from genomic DNA using (In1 F2 and In4 R primers), followed by cloning into pCR-Blunt II TOPO [Zero Blunt TM TOPO TM kit (Thermo Fisher)], and sequencing ≥3 clones from each cell line.CypA point mutant clones were screened via PCR amplification from genomic DNA using (In1 F and In4 R2 primers) followed by HpaI digestion.
Candidates were confirmed by PCR and sequencing from both genomic DNA and cDNA [using SuperScript TM III Reverse Transcriptase (Thermo Fisher)].Genomic DNA and mRNA were extracted using NucleoSpin Tissue and NucleoSpin RNA kits (Macherey-Nagel), respectively.

Viruses
All viruses were generated by transfection of 293T cells using polyethyleneimine (PolySciences).

Infection assays
Infectivity was measured in HeLa or HT1080 cells seeded in 96-well plates at 5 x 10 3 cells per well and inoculated with serial-dilutions of VSV-G pseudotyped GFP reporter viruses in the presence of 4μg ml -1 polybrene (Sigma-Aldrich).Where indicated, cyclosporine A (Sigma-Aldrich) was added to the cultures at the time of infection at 5μM.Infected cells (%GFP positive of viable cells) were enumerated by FACS analysis using an Attune NxT coupled to an Autosampler (Invitrogen).

CA-binding assay with HIV-1 CA tubes
CA(WT), CA(G89V), and CA(N57A) were expressed from pET3a in BL21-DE3 cells and purified as previously described (51)(52)(53).CA nanotubes were assembled in a high-ionic strength buffer (15 mM Tris-HCl, pH 8.0; 2 M NaCl) as described (54,55).Binding assays with HIV-1 CA nanotubes were performed as described (29).In brief, control and CypA mutant HT1080 cells were lysed by adding a passive lysis buffer (Promega) supplemented with protease inhibitor cocktail (Roche).NaCl concentration in lysates was adjusted to 2 M and lysates were centrifuged at 13,000 x g for 2 min at 4 C. Then the supernatant of the cell lysates was added to the preformed CA nanotubes and incubated at room temperature for 30 min.Following centrifugation at 13,000 x g for 2 min at 4 C, the supernatant was saved, and the pellet was washed three times with the high-ionic strength buffer.LDS Reducing Sample buffer (Thermo Scientific Chemicals) was added to both pulled-down and unbound fractions, and they were subjected to SDS-PAGE.The proteins of interest were detected by immunoblotting using respective antibodies.
Non-transfected cells, transfection reagent alone, and non-targeting siRNA were used as controls, no significant difference in viral infectivity or Mx2-restriction were observed for each of these controls, as such, for each experiment, only the non-targeting siRNA control is shown.24 hours after transfection, cells were trypsinized, diluted 1:2.5 and re-plated in 96-well plates and treated with doxycycline, followed by infection with GFP reporter viruses 36-48h later (See Figure S10B).

Statistical analyses
Statistical significance was determined using GraphPad software (two-way ANOVA).Statistical analyses for all figures are included in S1 File.A A A 1 3

S S E 1 1
A A A 1 3

S S E
To generate CypA mutant cell lines, we employed a CRISPR/Cas9-AAV-based gene engineering approach to target the PPIA locus (S2A Fig).CypA knock-out cells were generated by introduction of guide RNAs targeting intron 1 and exon 4, resulting in either a deletion of most of the coding sequence or a frameshift mutation that abrogated expression (S2B Fig).To generate point mutant cells, an AAV containing donor-DNA for homology-directed repair was introduced with D66N and/or R69H mutations (S2A and S2C Fig).Single cell clones with the desired mutation were confirmed by sequencing of both genomic DNA and mRNA.We then tested the ability of CypA in control and point mutant clones to bind HIV-1 CA in vitro (S3 Fig), and found that both CypAWT and CypAR69H, but not CypAD66N or CypAD66N/R69H pelleted with WT or N57A mutant CA, while CA(G89V) did not bind to CypA from any of the cell lines.
CsA treatment (S4 Fig).Infectivity and MX2 sensitivity of HIV-1A92E CA, HIV-1N74D CA, and HIV-1T210K CA in CypA -/-, CypAD66N, or CypAD66N/R69H cells also phenocopied CsA treatment in WT and CypAR69H cells.Furthermore, no additional effects of CsA treatment on HIV-1WT or CA mutant infection were observed in CypA knock-out or mutant cell lines (Fig 1B and S4 Fig).Collectively, similar to SIVmac, which does not bind cyclophilins, (Fig 1B and S4 Fig).These results indicate that unlike restriction of HIV-1, inhibition of other primate lentiviruses by MX2 is independent of CA-CypA interactions.Determinants for CA-specific effects of MX2 on lentivirus infection.We next sought to identify the determinants in MX2 that affect the CA-and CypA-specific sensitivity of lentiviruses to MX2 activity.We expressed a panel of (C-terminally myc tagged) MX2 mutants that altered the N-terminal CA-interacting domain, GTP binding and hydrolysis, or oligomerization (Fig 2) in HeLa and HT1080 cells and first determined their effect on HIV-1WT infection in the presence and absence of CsA.Murine leukemia virus (MLV) was included as an MX2-insensitive negative control.As expected (6, 7, 17, 21, 38), MX2 with a deletion of the N-terminal 25 amino acids (ΔN25), mutation of the N-terminal triple-arginine motif (RRR11-13AAA), or phosphomimetic S14D/S17D/S18D (SSS14,17,18DDD) did not inhibit HIV-1WT infection, while chimeric MX1 expressing the N-terminal 91 amino acids of MX2 (MX1MX2-NTD), and non-phosphorylatable S14A/S17A/S18A (SSS14,17,18AAA) had antiviral activity similar to WT MX2, that was abrogated in the presence of CsA HT1080, but not HeLa cells (Fig 3 and S5 Fig).Both the monomeric M574D Furthermore, MX2T151A failed to enhance HIV-1N57S CA infection in CypA -/-, huCypAD66N and CypAD66N/R69H cells.Sensitivity of HIV-2 to both WT and T151A mutant MX2 was similar in all cell lines, further demonstrating that CypA-interactions with HIV-2 CA do not affect MX2 sensitivity (S6 Fig).In 2022, Betancor, et al.(21) identified several serine and threonine residues of MX2 in addition to positions 14, 17, and 18 whose phosphorylation state affects the antiviral activity and substrate specificity of MX2.In particular, they found that MX2 T151 is phosphorylated, and demonstrated that a phosphomimetic MX2T151D exhibited increased antiviral activity against HIV-1WT and conferred antiviral activity against the MX2-resistant HIV-1N74D CA, HIV-1P90A CA, and HIV-1T210K CA.To determine whether the ability of the MX2T151A mutant to restrict HIV-1 infection in the absence of CypA-CA binding is due to the lack of a phosphate group at this residue or due to the inability to hydrolyze GTP, we assessed the antiviral activity of the phosphomimetic T151D MX2 mutant in HT1080 and HeLa cells (Fig4and S7 Fig).MX2T151D exhibited increased antiviral activity against HIV-1WT, and to a smaller extent against HIV-2 and SIVmac in both cell types.While the previous report indicated that MLV infection in U87-MG cells is sensitive to MX2T151D, we observed no restriction of MLV by this mutant.MX2T151D inhibited HIV-1N57S CA infection in both HeLa and HT1080 cells, and only partially rescued infectivity of this mutant in CsA-treated HT1080 cells (Fig 4 and S7 Fig).Finally, similar to MX2T151A, MX2T151D inhibited HIV-1WT infection in the presence of CsA as well as infection of HIV-1G89V CA, demonstrating that phosphorylation at MX2 residue T151 does not determine CA-CypA-dependent antiviral activity of MX2.Collectively, these results indicate that the inability to hydrolyze GTP abrogates the requirement for HIV-1 CA-CypA interactions in the antiviral activity of MX2.Effect of GTPase domain-CA interactions on MX2 activity in the absence of CypA-CA binding.One possibility explaining the ability of T151A/D mutants of MX2 to inhibit HIV-1 infection in the absence of CA-CypA interactions is that the lack of GTP hydrolysis facilitates interactions between the viral CA and the CA-binding site of the GTPase domain (39).To address this hypothesis, we constructed a number of MX2 mutants affecting GTPase activity (T151A), the Nterminal triple arginine motif (11AAA13) and/or the CA-binding residues in the GTPase domain [G184S, N260S, Q351E (SSE)] and assessed their antiviral activity (Fig 5 and S8 Fig).Mutation of the triple arginine motif abrogated the antiviral activity of MX2T151A against HIV-1WT, HIV-1G89V CA, HIV-2, and SIVmac.On the other hand, mutation of the CA-binding residues of the GTPase domain did not affect the antiviral activity of MX2T151A in the presence or absence of CsA against these viruses; in fact, the SSE/T151A mutant appeared to have enhanced antiviral activity as compared to WT or T151A MX2.Therefore, these CA-GTPase domain interactions do not mediate the antiviral activity of GTPase-deficient MX2.Interestingly however, mutation of GTPase domain-CA interacting residues did affect the enhancement of HIV-1N57S CA infection in CsAtreated HT1080 cells, almost fully restoring the ability of MX2T151A to rescue HIV-1N57S CA infection, suggesting that the lack of enhancement by MX2T151A under these conditions is at least partially mediated by GTPase domain-CA interactions.Next, we explored whether additional determinants affect the ability of the GTPasedeficient MX2 to restrict infection of primate lentiviruses in the absence of CA-CypA binding.We generated chimeric MX1MX2-NTD proteins with mutations corresponding to K131A (K83A) and T151A/D (T103A/D), as well as mutations in MX1 alone as a control, expressed them in HT1080 and HeLa cells, and assessed their antiviral activity (Fig 6 and S9 Fig).Inhibition of HIV-2 and SIVmac infection by chimeric MX1MX2-NTD was unaffected by all three mutations in both cell lines, while GTP binding was required for restriction by MX2 (Fig 3 and S5 Fig).The effect of these mutations on restriction of HIV-1WT was cell-type specific, as MX1MX2-NTD chimeras restricted HIV-1WT infection in the presence or absence of CsA in HeLa cells but did not restrict HIV-1WT in CsAtreated HT1080 cells.The effects on HIV-1G89V CA infection were also cell-type specific, while MX1MX2-NTD enhanced HIV-1G89V CA infection in both cell types, the K83A and T103A MX2 mutants modestly restricted HIV-1G89V CA infection.Furthermore, the T103D MX2 mutant had no effect on HIV-1G89V CA in HT1080 cells, while the opposite (no inhibition or enhancement by K83A and T103A, restriction by T103D) was observed in HeLa cells.Finally, HIV-1N57S CA infection was not inhibited by any MX1MX2-NTD chimeras in either cell type, and was rescued from the inhibitory effect of CsA treatment in HT1080 cells by all three GTPase MX1MX2-NTD mutants.The effect of GTPase mutations in MX1MX2-NTD chimeras on the HIV-1N57S CA mutant are in agreement with those shown in Figure5regarding the role of CA-interacting residues in the GTPase domain.However, the altered effects of GTP binding/hydrolysis mutant-MX1MX2-NTD chimeras on HIV-1WT, HIV-1G89V CA, HIV-2, and SIVmac infection as compared to the K131A, T151A, and T151D mutants of MX2 demonstrate that additional unknown determinants in the GTPase, stalk, or L4 domain affect interactions between MX2 and these viruses.Effects of Nup and NTR depletion on the antiviral activity ofGTPase-deficient MX2.Our previous work demonstrated Nups mediate the antiviral activity of MX2 in a CA and CypA-dependent manner (29).Since MX2T151A retains antiviral activity in the absence of CA-CypA interactions, we next sought to determine whether this mutation also affected Nup/NTR requirements for antiviral function.Using our previously validated siRNA library targeting Nups and NTRs (Fig 7A and (29)), HT1080 cells were transfected with siRNA before being split into replicated wells for MX2WT or MX2T151A induction (via doxycycline addition) followed by infection with HIV-1WT (S10 Fig).Knockdown of either RANBP2 or NUP153 abrogated the antiviral activity of both MX2WT and MX2T151A against HIV-1WT (Fig 7B-C).Furthermore, depletion of the Nup62 subcomplex, transmembrane, and nuclear basket Nups also had similar effects (eg.NUP62 depletion reduced MX2WT and MX2T151A activity, while NUP54, NUPL1, POM121, NDC1, NUP50, and TPR depletion either increased antiviral activity or had no effect), indicating that the T151A MX2 mutation does not completely alter MX2-Nup interactions.Moreover, the effects of NTR depletion were similar, suggesting that abrogation of GTPase activity does not affect MX2transportin interactions.However, a number of differences were evident in the Nup requirements for MX2WT and MX2T151A activity (eg.multiple components of the Nup107 and Nup93 subcomplexes reduced the antiviral activity of MX2WT, but not MX2T151A).Most notably, NUP88 and NUP214 depletion abolished the antiviral activity of MX2WT, but not MX2T151A.We next investigated the effects of Nup depletion on the MX2 sensitivity of HIV-1G89V CA.As we previously reported (29), the increase in HIV-1G89V CA infectivity exerted by MX2 is amplified by some Nup depletions (eg.NUP62, NUP88, NUP214, NUP153), and diminished by others (eg.NUP155, NUP54, RANBP2) (Fig 7D).As expected, a number of Nup depletions also altered the activity of MX2T151A against HIV-1G89V CA (Fig 7E).We observed no clear correlation between whether a knockdown affected enhancement of HIV-1G89V CA infection by MX2 and whether it caused loss of MX2T151A activity against HIV-1G89V CA.Depletion of transmembrane, cytoplasmic, and nuclear basket Nups, as well as NTRs had similar effects on the antiviral activity of MX2T151A against both HIV-1WT and HIV-1G89V CA (Fig 7C, E).Conversely, depletion of several Nups (SEC13, NUP107, NUP133, NUP205, NUP155, NUP52, NUPL1) affected restriction of HIV-1G89V CA but not HIV-1WT, while only NUP98 knockdown reduced antiviral activity against HIV-1WT but not HIV-1G89V ), retains antiviral activity in the absence of CA-CypA binding (Fig 3, S5 Fig, and S6 Fig).By generating cells containing endogenous mutations in CypA that affect CA recognition, we demonstrate that the effects of CypA (and CsA treatment) on HIV-1 and HIV-1 CA mutant infection and MX2 sensitivity are the direct result of interactions with CA, and do not appear to involve interactions between CypA and other cellular proteins (Fig 1, S4 Fig, S6 Fig).While the GTPase domain of MX2 has been found to directly bind the HIV-1 CA (39), we found that these interactions were not involved in the CypA-independent activity of GTPasedeficient MX2 (Fig 5 and S8 Fig).However, the antiviral activity of GTPase-deficient chimeric MX1MX2-NTD fusions did not recapitulate the phenotype of the T151A mutant (Fig 6 and S9 Fig), Interestingly, restriction of HIV-2, SIVmac, and SIVagmTAN by MX2 does not involve CA-CypA interactions ((29), Fig 1 and S4 Fig), and GTP hydrolysis is dispensable for inhibition of HIV-2 and SIVmac by both MX2 and MX1MX2-NTD fusions (Fig 3 and 6, S5 Fig and S9 Fig).
absence of CA-CypA interactions (Fig 4 and S7 Fig), indicating that the lack of phosphorylation of the T151A mutant does not explain its expanded substrate specificity.While the T151D MX2 . The plasmids generated expressed HA-TRIM5 N-terminus fused to the Cyclophilin A domain from owl monkey TRIMCyp (omkTRIMCyp), the human Cyclophilin A protein (huCypA), or the human Cyclophilin A protein with the D66N, R69H, or D66N/R69H (pigtail macaque TRIMCyp) mutations generated by overlap PCR with the primers indicated in S1 Table.
pAAV-PPIA donor plasmids for the generation of CypA point mutant cell lines (S2 Fig), a 1596bp region of the PPIA locus (bases 7161 to 8757 NCBI Reference Sequence: NG_029697.1)was synthesized as a gBlock TM fragment (IDT) and inserted into the pAAV packaging plasmid (Cell Bio Labs) using NheI and XhoI.The PAM site for crRNA 2 was mutated from TGG to TGA and for crRNA 3 form CCA to CTA.A silent HpaI site was introduced into intron 3 as shown in S2C Fig.The CypAR69H donor template was synthesized, then CypAD66N and CypAD66N/R69H donors were generated via overlap PCR with the primers indicated in S1 Table.
blot analysis of doxycycline-inducible MX2-myc and tubulin loading control.S6 Fig. Restriction of HIV-1 infection by GTPase-deficient MX2 in CypA mutant cells A) Infection of control and CypA-mutant HT1080 cell clones stably transduced with doxycyclineinducible myc-tagged MX2 (left) or MX2T151A (right) in the presence (open bars) or absence (filled bars) of doxycycline and presence or absence of CsA with the indicated GFP reporter viruses.Titers are represented as mean + sem of infectious units (IU) per pg of reverse transcriptase (RT), n≥12 technical replicates combined from five independent experiments.Statistical analysis in S1 File.B) Western blot analysis of doxycycline-inducible MX2-myc, CypA, and tubulin loading control in the indicated cell clones.S7 Fig. Phosphorylation at residue T151 does not determine CypA-independent MX2 activity.A) Infection HeLa cells stably transduced with doxycycline-inducible myc-tagged MX2 or MX2T151D in the presence (open bars) or absence (filled bars) of doxycycline and presence or absence of CsA with the indicated GFP reporter viruses.Titers are represented as mean + sem of infectious units (IU) per pg of reverse transcriptase (RT), n≥6 technical replicates combined from two-seven independent experiments.Statistical analysis in S1 File.B) Western blot analysis of doxycycline-inducible MX2-myc and tubulin loading control.S8 Fig. Restriction by GTPase-deficient MX2 in the absence of CA-CypA binding is not mediated by known CA-GTPase domain interactions.A) Infection of HeLa cells stably transduced with doxycycline-inducible myc-tagged MX2 with or without mutations in the N-terminal triple-arginine motif (11AAA13), T151A, or CA-binding residues in the GTPase domain (SSE) in the presence (open bars) or absence (filled bars) of doxycycline and presence or absence of CsA with the indicated GFP reporter viruses.Titers are represented as mean + sem of infectious units (IU) per pg of reverse transcriptase (RT), n=9 technical replicates combined from three independent experiments.Statistical analysis in S1 File.B) Western blot analysis of doxycycline-inducible MX2-myc and tubulin loading control.S9 Fig. Antiviral activity of GTPase-deficient chimeric MX1MX2-NTD proteins.A) Infection of HeLa cells stably transduced with doxycycline-inducible myc-tagged MX1 or MX1MX2-NTD with the indicated mutations in the presence (open bars) or absence (filled bars) of doxycycline and presence or absence of CsA with the indicated GFP reporter viruses.Titers are represented as mean + sem of infectious units (IU) per pg of reverse transcriptase (RT), n≥9 technical replicates combined from three-seven independent experiments.Statistical analysis in S1 File.B) Western blot analysis of doxycycline-inducible MX1MX2-NTD or MX1-myc and tubulin loading control.S10 Fig. Experimental design for Nup/NTR knockdown A) Experimental strategy to investigate the roles of Nups and NTRs in antiviral activity of MX2 and MX2T151A.For a detailed description, refer to the Materials and Methods.B) Western blot analysis of doxycycline-inducible MX2 and MX2T151A and tubulin loading control.