# Deciphering lentiviral Vpr/Vpx determinants required for HUSH and SAMHD1 antagonism highlights the molecular plasticity of these evolutionary conflicts

Pauline Larrous, Cassandre Garnier, Marina Morel, Michael Martin, Karima Zarrouk, Sarah Maesen, Roy Matkovic, Andrea Cimarelli, Lucie Etienne,

Florence Margottin-Goguet

► To cite this version:

Pauline Larrous, Cassandre Garnier, Marina Morel, Michael Martin, Karima Zarrouk, et al. Deciphering lentiviral Vpr/Vpx determinants required for HUSH and SAMHD1 antagonism highlights the molecular plasticity of these evolutionary conflicts. Journal of Virology, 2025, 99 (5), 10.1128/jvi.00198-25. hal-05367719

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1

2 Deciphering lentiviral Vpr/Vpx determinants required for HUSH and

3 SAMHD1 antagonism highlights the molecular plasticity of these

4 evolutionary conflicts

5

6

7 Pauline Larrous¹, Cassandre Garnier¹, Marina Morel¹, Michael M. Martin¹, Karima Zarrouk¹,

8 Sarah Maesen², Roy Matkovic¹, Andrea Cimarelli², Lucie Etienne²*, Florence Margottin-

9 Goguet¹*

10

11 ¹Institut Cochin, Université Paris Cité, INSERM U1016, CNRS UMR8104, Paris, France

12 ²CIRI, Centre International de Recherche en Infectiologie, Inserm U1111, Université Claude

13 Bernard Lyon 1, CNRS UMR5308, Ecole Normale Supérieure de Lyon, Univ Lyon, Lyon,

14 France

15

16

17 * Corresponding authors:

18

19 - Florence Margottin-Goguet (22 rue Méchain, 75014 Paris, France; tel: +33 1 40 51 66

20 16; florence.margottin-goguet@inserm.fr)

21

22 - Lucie Etienne (46 Allée d'Italie, 69364 Lyon Cedex 07, France ; +33 (0)4 72 72 86 96 ;

23 lucie.etienne@ens-lyon.fr)

24
## 25 ABSTRACT

26 SAMHD1 and the HUSH complex constitute two blocks during primate lentivirus infection,
27 the first by limiting reverse transcription and the second by inhibiting proviral expression. Vpr
28 and Vpx of specific lentiviral lineages have evolved to antagonize these antiviral proteins.
29 While the antagonism of SAMHD1 has been well characterized, the evolutionary and
30 molecular determinants of the antagonism against HUSH are unknown. We used chimeric
31 Vpr proteins between SIVagm.Ver and SIVagm.Gri lentiviruses infecting two African green
32 monkey species, to investigate viral determinants involved in HUSH and SAMHD1
33 antagonisms. We found that different interfaces of closely related Vpr proteins are engaged to
34 degrade different SAMHD1 haplotypes. In addition, we identified distinct viral determinants
35 in SIVagm.Ver Vpr for SAMHD1 and HUSH degradation. The substitution of one residue in
36 SIVagm.Gri Vpr is sufficient to gain the capacity to degrade SAMHD1, while the substitution
37 of α-helix-3 confers HUSH antagonism. We also found that Vpx proteins of HIV-2 from
38 people living with HIV have different abilities to degrade HUSH. These phenotypes rely on
39 small changes in either the N or C terminal part of Vpx, depending on the context. On the host
40 side, we found that HIV-2 and SIVsmm Vpx degrading HUSH from human and vervet
41 monkey cells cannot not degrade HUSH in owl monkey cells, suggesting some host species-
42 specificity.

43 Altogether, we highlight the molecular plasticity and constraints of viral proteins to adapt to
44 host restrictions. HUSH, like SAMHD1, may have been engaged in ancient and more recent
45 coevolution with lentiviruses and a player in viral fitness.
46

## 47 IMPORTANCE

48 Antiviral host proteins, the so-called restriction factors, block lentiviruses at different steps of
49 their viral life cycle. In return, primate lentiviruses may counteract these immune proteins to
50 efficiently spread *in vivo*. HIV-2 and some SIVs, but not HIV-1, inactivate SAMHD1 and
51 HUSH, two host antiviral proteins, thanks to their Vpx or Vpr viral proteins. First, we
52 uncovered here viral determinants involved in the function of closely related Vpr proteins
53 from SIVs of African green monkeys and of HIV-2 Vpx alleles from people living with HIV-
54 2. We show how these small viral proteins differently adapted to SAMHD1 polymorphism or
55 to HUSH restriction and highlight their molecular plasticity. Finally, the capacity of divergent
56 lentiviral proteins, including HIV-2 Vpx, to induce the degradation of HUSH depends on the
57 cell/host species. Altogether, our results suggest that HUSH has been engaged in a molecular
58 arms-race along evolution, and therefore is a key player in host-pathogens interaction.
59

60 **KEYWORDS**

61 HIV, SIV, restriction factors, coevolution, HUSH, SAMHD1, viral antagonism

62
63 INTRODUCTION

64 Host restriction factors are antiviral proteins from the cell autonomous immunity that have
65 been engaged in an evolutionary arms-race with the pathogenic viruses they have been
66 fighting for millions of years (1, 2). They further represent molecular barriers to cross-species
67 transmission of viruses (3-6). When transmitted to humans, the lentiviruses SIVcpz and
68 SIVgor (from chimpanzees and gorillas, respectively) and SIVsmm (from sooty mangabeys)
69 gave rise to HIV-1 and HIV-2, respectively. The different lentiviral lineages share a similar
70 genomic organization, but differ in their set of accessory genes, which produce proteins
71 largely dedicated to the counteraction of restriction factors and which have strongly evolved
72 during lentiviral cross-species transmissions (6, 7). Determining the exact molecular
73 residues/interfaces underlying these conflicts is therefore a major objective to better
74 understand HIV-cell interactions and determinants of virus spillover.
75

76 All extant primate lentiviruses, including SIVagm (infecting African green monkeys), encode
77 Vpr, which induces host G2/M cell cycle arrest (8-10). However, despite its importance in the
78 dissemination and pathogenesis of SIVsmm (11), Vpx is found in only two of the eight major
79 lineages of primate lentiviruses, HIV-2/SIVmac/SIVsmm (infecting humans, macaques and
80 sooty mangabeys) and SIVrcm/mnd2 (infecting red-capped mangabeys and mandrills). Vpr
81 and vpx genes are the results of duplication and recombination events of a precursor gene
82 (reviewed in (12)). The encoded proteins share similarities in size (about 100 amino acids),
83 structure (a N-terminal tail, 3 α-helices and a C-terminal tail) and functions. Nonetheless,
84 they also present highly variable regions: VR1 upstream of helix 1, VR2 across the end of
85 helix 2 and the beginning of helix 3, and VR3, which overlaps the C-terminal tail. In viral
86 lineages that encode both Vpr and Vpx, Vpx induces the proteasomal degradation of the host
87 restriction factor SAMHD1 (SAM and HD domain-containing protein 1), while in some other
88 lineages that do not encode Vpx, such as SIVagm, the Vpr protein performs this function (13-
89 15). Associated phylogenetic analyses showed that an ancestral Vpr protein acquired the anti-
90 SAMHD1 activity prior to the molecular events that gave birth to Vpx (15). SAMHD1 is a
91 626 amino acid dNTPase that blocks viral DNA synthesis by lowering the pool of nucleotides
92 in macrophages and quiescent CD4+ T cells (16, 17). By degrading SAMHD1, Vpx/Vpr
93 proteins enable the virus to bypass a reverse transcription block. In this process, Vpx/Vpr
94 directly binds SAMHD1 and bridges SAMHD1 to the DCAF1 adaptor of a Cullin4A-based
95 ubiquitin ligase (13, 14, 18-21).
96 How Vpx interacts with SAMHD1 in a virus-host species-specific manner has been
extensively studied. Strikingly, search for host determinants revealed that HIV-2/SIVsmm Vpx targets the C-terminus of SAMHD1, while SIVmnd2 and SIVrcm Vpx recognize its N-terminus (22-24). The divergence in SAMHD1 recognition is further witnessed by the presence of sites under positive selection during primate evolution in both N- and C-terminal domains of the protein (15, 22, 25). These site-specific adaptations in SAMHD1 are the results of host escape from viral antagonism, characteristic of a molecular virus-host arms-race.

The resolution of crystal structures and functional studies have allowed the identification of the interfaces between Vpx from different lineages and SAMHD1 (23, 24). In particular, a cluster of residues in the Vpx α-helix 2 of SIVmnd2 are in contact with SAMHD1, while these amino-acids are not involved in the case of Vpx from SIVsmm (23, 24).

By studying the coevolution between African green monkeys (AGMs) and their SIVs, Spragg and Emerman showed that SAMHD1 antagonism is crucial for viral fitness (26). AGMs comprise at least four closely-related species : *Chlorocebus tantalus*, *sabaeus*, *aethiops* (Grivet) and *pygerythrus* (Vervet). Each lineage of SIVagm, responsible of the natural infection of each species, has evolved to antagonize distinct SAMHD1 haplotypes through the use of Vpr (26). More precisely, among the seven SAMHD1 haplotypes identified in the AGMs, haplotype IV, but not V, is degraded by SIVagm.Ver Vpr, while the opposite is found for SIVagm.Gri Vpr; haplotype III is resistant to both Vprs, but is sensitive to SIVagm.Sab Vpr (26).

In addition to SAMHD1, HIV-2/SIVsmm Vpx and the Vpr from specific lineages can induce the degradation of the human HUSH complex (27, 28). The HUSH complex is composed of TASOR, MPP8 and periphilin and contributes to the silencing of cellular genes and retroelements with the help of MORC2(29). Due to HUSH degradation, Vpx/Vpr proteins favor viral expression in a model of HIV-1 latency (27, 28). Functional and evolutionary studies led us to conclude that HUSH antagonism is likely an ancient function of primate lentiviruses that preceded the birth of Vpx and SAMHD1 antagonism (15). In addition, HUSH antagonism appeared lentiviral species-specific, with only some Vpx/Vpr proteins degrading human HUSH. Whether lentiviral species-specificity is accompanied by host species-specificity, in line with host-virus competition along evolution, has not been investigated yet in the case of HUSH.

Here, we took advantage of the lentiviral-host specificity within the AGM lineage, both for SAMHD1 and HUSH antagonism, to identify viral determinants at stake. We found that the closely related Vpr proteins use different viral determinants to degrade different SAMHD1
haplotypes, highlighting the molecular plasticity and adaptation of the virus to the host. In addition, viral determinants against HUSH are different from those against SAMHD1. We further studied Vpx proteins of HIV-2 from people living with HIV-2 (PLWH-2), which were competent for SAMHD1 degradation (30). We found that some Vpx proteins can induce HUSH degradation, while others cannot, and this diversity seems independent of the corresponding status of the viremia. Depending on the Vpx, restoration of the capacity to induce HUSH degradation was obtained by changing either the N-terminal part or the C-terminal part of the viral protein.

Finally, we describe one evidence of host-species specificity with HIV-2/SIVsmm Vpx unable to counteract HUSH in New World monkey cells. Altogether, our results suggest the existence of a dynamic interplay between HUSH, SAMHD1 and primate lentiviruses along evolution.

143

## 144 MATERIALS AND METHODS

### 145 Plasmids

Vpr SIVagm.ver9063 (KF741096), Vpr SIVagm.gri677 (sequence as the one used in (26)), Vpr SIVagm.Tan1 (U58991) and Vpr SIVagm.Sab1 (S46351) chimera 1 to 10 (sup Fig 1), Vpx proteins of HIV-2 from PLWH-2 and Vpx chimeras have been synthesized after codon-optimisation and subcloned into the pAS1b vector (pAS1b-HA) to get HA-epitope-tagged proteins (HA at the N-terminus). Vpr SIVagm.Tan1 is also expressed in pCDNA3-3xFlag vector with a Flag epitope at the N-terminus. The mutants of Vpr were produced by site-directed mutagenesis according to Phusion polymerase manufacture guide (Thermofisher) or CloneAmp HiFi polymerase manufacture guide (Takkara) using Vpr SIVagm.ver9063 or chimera 8 or 9 in the pAS1B vector as templates. Lentiviral proteins Vpx HIV-2 Gh (P18045.1), Vpx SIVsmm (P19508.1), Vpx SIVmnd2.GAx14 (AAK82846.1), Vpx SIVrcm.NG411 (AAK69676.1) and Vpx SIVrcm.Gab1 (AAM34564.1) are also expressed from the pAS1b vector (HA tag at the N-terminus). Human TASOR (NP_001106207.1) and Owl monkey TASOR (NCBI References Sequences: XP_012316204) are expressed from vectors pLenti-Flag and pCMV6-Flag respectively, in fusion with the myc-DDK epitope at the C-terminus. Constructs expressing Haplotypes III, IV and V of SAMHD1 from AGMs are gifts from M. Emerman and are expressed with a HA epitope at the C-terminus from the pLPCX vector (KF741043, KF741044 and KF741045).

163

### 164 Cell culture
Cells were tested regularly for mycoplasma contaminations; experiments were only performed on non-contaminated cells. ATCC-purchased HeLa (CCL-2), VERO (CCL-81), HEK293T (CLR-3216), HEK293FT (293T cells optimized for VLP production, gift from N. Manel) and OMK cells were cultivated in media DMEM (Thermofisher) containing 10% fetal bovine serum (FBS, Eurobio), 1,000 units/mL penicillin, 1,000µg/mL streptomycin (Life Technologies). J-Lat A1 (gift from E. Verdin) were cultivated in media RPMI (Thermofisher) supplemented as described for the DMEM medium and, in addition, 2mM glutamine (Life Technologies). HEK293T stable cell lines expressing the haplotypes III, IV and V of AGM SAMHD1 were generated by transduction of VLP containing pLPCX-Hap III, pLPCX-Hap IV and pLPCX-Hap V and cultivated four days before puromycin selection.

## siRNA treatment

siRNA transfections were performed with DharmaFECT1 (Dharmacon, GE Lifesciences). The final concentration for all the siRNA was 40nM. The following siRNA were purchased: siTASOR: SASI_Hs02_00325516 (Sigma Aldrich); siDCAF1: J-021119-10-0005 (Dharmacon) and the non-targeting control siRNA: MISSION siRNA Universal Negative Control 1, SIC001 (Sigma Aldrich).

## Virus-Like-Particle production, delivery and transduction

VLPs were produced in HEK293FT cells by co-transfection by the calcium-phosphate co-precipitation method of VSV-G plasmid (3µg), SIV3+ ΔVpr ΔVpx vectors (8µg) and 8µg of pAS1b-HA-Vpr (or chimeric proteins or point mutants) or pAS1b-HA-Vpx or pAS1b-HA (empty) and in some experiments with a transfer gene pGAE1.0 (SIVmac-CMV-GFP) used as a reporter gene. SIV3+ ΔVpr ΔVpx packaging vector is a gift from N. Landau and is described in Gramberg *et al* (31). VLPs used for the establishment of the HEK293T stable cell lines expressing HA-SAMHD1 (agm) were produced with VSV-G plasmid (3µg), pHIT60 MLV packaging vector (8µg) and 8µg of pLPCX vector expressing Hap III or Hap IV or Hap V. In both cases, 3.10⁶ cells were plated in 10cm culture dishes the day prior transfection. Cell culture medium was collected 48h after transfection and filtered through 0.45µm pores filters. For SAMHD1 VLP, 10mM NaBu were added 24h after transfection and the cells washed at the end of the day. VLP were concentrated by sucrose gradient and ultracentrifugation (1h30 at 100,000g). The incorporation of the viral proteins was assessed by western blot and a quantification of the level of HIV-2 capsid (P27) and HA-Vpr was performed to deliver the same quantity of viral proteins. J-Lat A1 cells were treated with VLP
199

200

201

for 7h in reduced medium prior to overnight TNFα (1ng/mL) treatment. VERO and OMK cells were plated in 12-well dishes at a density of 3.10⁵ cells and transduced the day after in reduced medium, cells were harvested the day after transduction.

202

203

### Flow cytometric analyses

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205

206

207

208

J-Lat A1 cells were collected and resuspended in PBS-EDTA (0,5mM). Data were collected and analyzed with BD Accuri C6 cytometer or Attune and software CFlow Plus or FlowJo. At least 10,000 events in P1 were collected, the GFP-positive population was determined using untreated J-Lat A1 cells as their level of GFP expression is low. The same gate was maintained for all conditions and analysis were performed overall GFP-positive population.

209

210

### SAMHD1 degradation assay

211

212

213

214

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216

217

218

219

HEK293T Hap III, Hap IV and Hap V were plated in 12-well dishes at 1,5.10⁵ cells and transfected the following day using the calcium-phosphate co-precipitation method. Different amounts of the pAS1B vector expressing the different Vpr proteins (1 to 2µg) were transfected to get the same level of expression of the viral proteins (with adjustment to get the same level of total DNA per condition). Cells were harvested 48h post-transfection for western blot analysis. Lysis of the cells was performed in 100µL of RIPAC buffer (50mM Tris-HCl pH7.5, 150mM NaCl, 10% Glycerol, 2mM EDTA, 0.5% NP40, 0.1% SDS) containing an anti-protease cocktail (A32965, Thermofisher), lysates were centrifuged at 16,000g for 10min to remove cell debris.

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221

### Cell fractionation

222

223

224

225

226

227

228

229

230

231

All immunoprecipitation experiments are performed in the nuclear fraction of HeLa cells. Cells grown in 10cm dishes were washed with cold Dulbecco's PBS 1x (ThermoFisher). After trypsinization (Thermofisher), cells were recovered in 1,5mL tubes and washed once with ice-cold PBS. After 4 min of centrifugation at 400xg, 1mL of cytoplasmic lysis buffer (10 mM TRIS-HCl pH7.5, 10 mM NaCl, 3 mM MgCl2, and 0.5% IGEPAL® CA-630 (I8896-100ML Merck)) was added on the cell pellet and resuspended pellet was incubated on ice for 5 min. Cells were then centrifuged at 300g for 4 min at 4 °C and the supernatant was saved for cytoplasmic fraction. The pellet was washed with 1mL of cytoplasmic lysis buffer and re-centrifuged at 300g for 4 min at 4 °C. Finally, the nuclear pellet was lysed with 300µL of RIPA buffer.

232
233 **Immunoprecipitation assay, western blot procedure and antibodies**

234 For HA-Vpr (AGM WT or chimeric proteins) and TASOR-Flag immunoprecipitations, HeLa
235 cells were plated at respectively 2,5.10⁶ cells in 10cm dishes and co-transfected by the
236 calcium-phosphate co-precipitation method with pAS1b-HA or pAS1b-HA-Vpx or Vpr
237 (SIVagm WT or chimeric proteins) and plenti-TASOR-FLAG or pCMV6-TASOR-FLAG.
238 Cells were harvested 48h post-transfection for western blot analysis. Lysis of the cells was
239 performed in 700µL of RIPA buffer (50mM Tris-HCl pH7.5, 150mM NaCl, 10% Glycerol,
240 2mM EDTA, 0.5% NP40) containing an anti-protease cocktail (A32965, Thermofisher) and
241 spun at 16,000g for 10min to remove cell debris. 500µg of cell lysates were incubated with
242 pre-washed EZview Red ANTI-HA or FlagM2 affinity Gel Beads (E6779 and F2426, Merck)
243 at 4 °C under overnight rotation. After three washes in wash buffer (50mM Tris-HCl pH7.5,
244 150mM NaCl), immunocomplexes were eluted with Laemmli buffer 1X with 20mM DTT and
245 were separated by SDS-PAGE (Bolt Bis-Tris, 4-12%, Life Technologies). Following transfer
246 onto PVDF membranes, proteins were revealed by immunoblot and signals were acquired
247 with Fusion FX (Vilber Lourmat). The following antibodies, with their respective dilution in
248 5% skimmed milk in PBS-tween 0.1% were used: anti-HA-HRP (3F10) (N°12013819001,
249 Roche) 1/10,000; anti-FLAG-HRP (A-8592, lot 61K9220, Sigma) 1/10,000; anti-HA (HA-7,
250 H3663, lot 066M4837V, Merck) 1/1,000; anti-Flag M2 (F1804-200UG- lot SLCD3990,
251 Merck) 1/1,000; anti-TASOR (HPA006735, lots A106822, C119001, Merck) 1/1,000; anti-
252 DCAF1 (11612-1-AP, ProteinTech) 1/1000; Anti-p27/p55 and anti-P24 were provided by the
253 NIH AIDS research and reference reagent program (ref ARP392/393) 1/1000; anti-βActin
254 (AC40, A3853, Merck) 1/1000; anti-GAPDH (6C5, SC-32233, Santa Cruz) 1/1,000. All
255 HRP-conjugated secondary antibodies, anti-mouse (31430, lot VF297958, Thermofisher) and
256 anti-rabbit (31460, lots VC297287, UK293475 Thermofisher), were used at a 1/20,000
257 dilution before reaction with Immobilon Classico (WBLUC0500, Merck Millipore) or Forte
258 (WBLUF0100, Merck Millipore) Western HRP.
259

259

260 **Immunofluorescence assay**

261 HeLa cells were cultivated on glass side and transfected as explained before. Cells were fixed
262 with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton for 15 min.
263 Blocking step was performed with 2% bovine serum albumin solution for 1h at room
264 temperature. Cells were incubated with antibody against anti-hemagglutinin mouse IgG
265 monoclonal conjugate Alexa Fluor 488 conjugated (Invitrogen) for 1h. The cells were washed
266 and the nuclei counter stained with 40-6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich)
267 for 20 min. The coverslip cells were mounted with Prolong gold/diamond antifade reagent (Invitrogen). Immunofluorescence images were captured by using a Leica DMI6000 confocal microscope at the IMAG'IC core facility.

270

271

272 **RESULTS**

273

274 To identify viral determinants involved in SAMHD1 and HUSH antagonism, we leveraged the lentiviral-host specificity within the SIV-AGM lineage. Among the seven SAMHD1 haplotypes identified in the AGM population (26), we chose to study three haplotypes (III, IV and V) that present no more than four amino acid differences and that have been described as: abundant in the Vervet but very rare in the Grivet AGM species (haplotype IV), or abundant in the Grivet and absent in the Vervet AGM species (haplotype V), or absent/rare in the Grivet/Vervet AGM species (haplotype III) (Fig. 1A). The ability of Vpr proteins from SIVagm viruses to degrade the different SAMHD1 haplotypes was then examined (Fig. 1A and 1B). To this end, 293T stable cell lines encoding the different HA-tagged SAMHD1 haplotypes were first established and Vpr was then expressed ectopically by transient DNA transfection (primary sequences of Vpr proteins in Sup Fig. 1). Of note, the sequence of SIVagm.Gri Vpr (SIVagm.grv677 Vpr) harbors two amino-acid changes (A2T and R102G) compared to the sequence of the original protein (NCBI RefSeq NP_054371.1), which were acquired after virus isolation (26, 32). The sequence of SIVagm.Ver Vpr is from SIVagm.Ver9063 (GenBank KF741096.1). Under these conditions, the Vpr protein derived from the SIVagm.Ver9063 was able to induce the degradation of SAMHD1 haplotype IV, but not haplotype V (Fig. 1B, quantification on the right). Conversely, the Vpr protein derived from the SIVagm.grv677 led to the degradation of SAMHD1 haplotype V but not haplotype IV (Fig. 1B). As previously shown by Spragg and Emerman (26), neither of the two Vpr proteins induced the degradation of SAMHD1 haplotype III, which is predominantly found in Sabaeus AGM species (Fig. 1B). The observed specificities of Vpr proteins could not be explained by major subcellular localisation differences, because both Vprs exhibited similar distribution in the nucleus and the cytoplasm, following immunofluorescence and biochemical fractionation experiments (Fig. 1C and 1D). However, the specificities could be the result of post-translational modifications, as at least two bands were revealed in different proportions for SIVagm.Ver Vpr and SIVagm.Gri Vpr, but this was not further investigated (Fig. 1B and 1D). Overall, phenotypes of SAMHD1 degradation suggest the existence of
301 specific interactions between SAMHD1 haplotypes present in a given species and the Vpr
302 protein from the SIVagm infecting the same species.

303 SIVagm.Ver and SIVagm.Gri Vpr proteins share 69% of identity at the amino acid
304 level. To decipher the viral determinants involved in SAMHD1 antagonism, we constructed
305 Vpr protein chimeras by exchanging their different α-helices, N- and C-termini domains (NtD
306 and CtD) (Fig. 1E, Sup Fig. 1 for sequences). Some chimeras harbored a dimeric form in
307 addition to the monomeric form, but all were able to bind DCAF1 (Fig. 1F). Chimera 6 was
308 hardly present as a monomer, possibly impacting its activity (Fig. 1F). All chimeras able to
309 induce SAMHD1 haplotype IV degradation in human cells contained the CtD of SIVagm.Ver
310 Vpr (chimeras 1, 4, 5, 7) (Fig. 2A). The replacement of the CtD from SIVagm.Gri by the CtD
311 from SIVagm.Ver (chimera 9) conferred the SIVagm.Gri Vpr the ability to induce SAMHD1
312 haplotype IV degradation (gain-of-function, Fig. 2A). These results suggest that the integrity
313 of the C-terminal domain of SIVagm.Ver Vpr is a critical determinant for SAMHD1
314 haplotype IV degradation. Furthermore, the CtD of SIVagm.Tan Vpr was also able to confer
315 the SIVagm.Gri protein (chimera 10) the ability to degrade SAMHD1 haplotype IV (Fig. 2B).
316 Therefore, SIVagm Vpr proteins that target the C-terminus of SAMHD1 (SIVagm.Tan and
317 SIVagm.Ver Vpr (26)) rely on C-terminal Vpr determinants to induce SAMHD1 haplotype
318 IV degradation.

319 None of the viral chimeras led to consistent degradation of SAMHD1 haplotype V,
320 suggesting that determinants throughout the viral protein, or the protein's complete
321 conformation, are essential for this activity (Fig. 2C). Despite all chimeras were able to bind
322 DCAF1, we cannot exclude that the lack of degradation might result from a global structural
323 defect. Therefore, only chimeras able to degrade either SAMHD1 haplotype IV (1,4,5,7,9,10)
324 or TASOR (1,4,5,8, see below) were taken into account to draw conclusions. With this in
325 mind, our results show that different viral interfaces in SIVagm.Ver Vpr and SIVagm.Gri Vpr
326 are involved in the degradation of SAMHD1 haplotypes IV and V, respectively.

327
328 We then used the same Vpr chimeras to identify the viral determinants involved in the
329 degradation of TASOR, the core component of the HUSH complex (33). First, we assayed the
330 degradation of endogenous TASOR in AGM Vervet cells (VERO cells). Vpr proteins were
331 incorporated into Viral-Like Particles (VLPs) and then delivered in VERO cells. Vpr
332 incorporation into VLPs was quantified by Western-blot, and VLPs quantities were adjusted
333 to deliver similar amounts of viral proteins into cells (Fig. 3A). SIVagm.Ver, SIVagm.Sab
334 and SIVagm.Tan Vpr proteins were able to induce the degradation of endogenous TASOR in
Vervet cells, in contrast to the SIVagm.Gri Vpr (Fig. 3B). Of note, our TASOR antibody detected two bands in VERO cells, but subsequent TASOR siRNA experiment suggested that only the lower band was indeed corresponding to TASOR (Fig. 3B, right). As previously described, similar phenotypes of TASOR degradation were found in human J-Lat A1 cells (Fig. 3C), a Jurkat T-cell line derivative that harbors a latent HIV-1 mini-genome expressing GFP under the control of the proviral LTR promoter (27, 34). In this HIV-1 latency model, TASOR degradation correlated with an increase of the percentage of GFP-positive cells, indicative of the reactivation of the latent provirus (Fig. 3D). Chimeras were further tested in both cell types, VERO and human cells (Fig. 4). All chimeras were well incorporated into VLPs, except chimera 2 (Fig. 4A). Degradation phenotypes were the same in Vero and J-Lat A1 cells: all the Vpr constructs that induced TASOR degradation contained the SIVagm.Ver Vpr α-helix 3 (chimeras 1, 4, 5 and 8) (Fig. 4B for Vero cells and 4C for human J-Lat A1 cells). In particular, chimera 8, which harbors the SIVagm.Ver α-helix 3 in a SIVagm.Gri Vpr background, was competent for TASOR degradation (gain-of-function), indicating that the SIVagm.Ver Vpr α-helix 3 region is a key viral determinant for TASOR degradation. As expected, TASOR degradation correlated with reactivation in the J-Lat-A1 model (Fig. 4D). Altogether, the C-terminal domain of SIVagm.Ver Vpr appears as a critical determinant for SAMHD1 haplotype IV degradation, while α-helix 3 is a key determinant for TASOR degradation.

To further delineate key residues, we performed sequence analyses of the viral proteins in the two regions. We first searched for potential “loss of function” mutations, which would impair SAMHD1 haplotype IV degradation, in the few residues that differ between the CtD of SIVagm.Ver and SIVagm.Gri Vpr proteins (Fig. 5A). We made the corresponding Vpr mutants by changing residues in the CtD of chimera 9, thus producing chimera 9 Q94G, E97S and RANRA-APPP (with RANRA residues at position 110 changed to APPP). We also made the R102G mutant directly in the SIVagm.Ver Vpr. Of note, D104 and D119 were not changed, because they are both present in the phenotypically different Vpr. The resulting proteins were all able to induce SAMHD1 haplotype IV degradation, except SIVagm.Ver Vpr R102G (Fig. 5B). Furthermore, the reciprocal G102R change in SIVagm.Gri Vpr restored its ability to induce SAMHD1 haplotype IV degradation, highlighting R102 as a key residue for this activity (Fig. 5C). Of note, the G102R substitution allowed to recover the original sequence of SIVagm.Gri Vpr; the original protein is now
368 notified with an asterisk throughout the manuscript (i.e., SIVagm.grv677* Vpr). Results are
369 summarized in Figure 5D.

370 Similarly, we searched for potential “loss of function” mutations, which would impair
371 TASOR degradation, by analyzing differences in α-helix 3 (Fig. 6A). Six residues that were
372 different between SIVagm.Ver and SIVagm.Gri Vpr proteins in the α-helix 3 were changed
373 two by two. Incorporation of the corresponding viral proteins into VLPs was checked (Fig.
374 6B). All the corresponding mutants in the SIVagm.Ver Vpr background retained the capacity
375 to degrade human TASOR and to reactivate HIV-1 in the J-Lat A1 model (Fig. 6C and 6D).
376 Because several residues in α-helix 3 of Vpr/Vpx proteins are required for DCAF1 binding
377 (19, 23, 24), we wondered whether the defect in TASOR degradation could result from a
378 defect in DCAF1 binding. However, we found that SIVagm.Ver and SIVagm.Gri Vpr
379 proteins both bound DCAF1 suggesting that differences in TASOR degradation are not linked
380 to DCAF1 binding (Fig. 1F or 6E). Interestingly, exogenously-expressed Flag-tagged human
381 TASOR seemed to better interact with SIVagm.Ver Vpr than with SIVagm.Gri Vpr (Fig. 6E).
382 Vpr differences in TASOR degradation could then result from differences in TASOR binding.
383 Mutations two by two in α-helix 3 did not impair DCAF1 or TASOR-Flag binding in
384 agreement with degradation assays (Fig. 6E). Thus, α-helix 3 appears essential for TASOR
degradation, but we could not further narrow-down the specific residues.
385

386 Overall, recurrent adaptation cycles to antagonize restriction factors during lentiviral
387 evolution have selected different determinants (summarized in Fig. 5D).

388

389 We next explored the specificity and determinants against SAMHD1 and TASOR
390 within the HIV-2 clade, which is a single lentiviral lineage as opposed to SIVagms.
391 Specifically, we tested whether Vpx from different HIV-2 strains could have different
392 abilities to induce TASOR degradation. We tested a panel of seven HIV-2s from the A or B
393 pandemic groups (sequences Sup Fig. 1): control Vpx from Ghana-1 (group A), Rod (group
394 A) and JK (group B) strains and four Vpx alleles of HIV-2 derived from PLWH-2, which
395 were selected from a study by Yu et al as able to induce the degradation of SAMHD1 (30).
396 Two alleles were from effective controllers (EC: high CD4+T cells and undetectable viral
397 load for several years) and two from non-controllers (NC: low CD4 count and high viral load)
398 (30). Vpx from Ghana-1, Rod and JK strains, one NC Vpx (RH2-1-D8, group A) and one EC
399 Vpx (RH2-135C1, group A) were able to induce TASOR degradation in human J-Lat A1 cells
400 after VLP delivery and subsequently to reactivate HIV-1 in the J-Lat A1 model (Fig. 7). One
401 NC Vpx (RH2-7B3, group A) and one EC Vpx (RH2-22-1B4, group B) were impaired in
402 their ability to induce TASOR degradation and reactivation of the latent virus (Fig. 7, strongly
403 impaired for B3, poorly for B4). Therefore, the efficiency of Vpx-mediated TASOR
404 degradation and HIV reactivation was variable within the HIV-2 lineage and did not correlate
405 with the potency of viral control in PLWH-2. We further took advantage of these phenotypic
406 differences and constructed chimera to identify Vpx determinants. In particular, we used NC
407 Vpx RH2-1-D (competent for TASOR degradation) and NC Vpx RH2-7B3 (non competent).
408 The two share 84.8 % identity, with most differences (highlighted in bold in Fig. 8A) in two
409 regions at the N-ter (N-ter tail + first part of helix-1) and the C-ter (helix 3 + C-ter tail). We
410 found that the replacement of the N-ter region (chimera D8B3), but not the C-ter (chimera
411 B3D8B3), from Vpx RH2-7B3 by RH2-1-D8 led to TASOR degradation and reactivation of
412 latent HIV-1 (gain-of-function, Fig. 8; C: incorporation, D: degradation, E: reactivation),
413 showing that RH2-1-D8 Vpx N-ter has key determinants in TASOR antagonism. Yet, in the
414 context of another chimera (B4D8B4, Fig. 9), we revealed that the C-ter region was this time
415 important for improving TASOR degradation efficiency (Figure 9). Overall, the capacity to
416 trigger TASOR degradation is context dependent and relies on N-terminal and C-terminal
417 regions of the viral protein.
418

419 This study and previous work (27, 28) showed differences between Vpr/Vpx proteins
420 from various lentiviral lineages to counteract human TASOR ("lentiviral lineage-specificity").
421 To complete the virus-host heterologous assays, we next tested the ability of lentiviral
422 SIVagm Vpr proteins to degrade TASOR from divergent primate host species, using cells
423 from New World monkeys (NWMs; owl monkey *Aotus trivirgatus* (aotTri) kidney OMK
424 cells) in addition to cells used up to now from hominoids (human J-Lat A1 cells) and Old
425 World monkeys (OWMs; AGM Vervet Vero cells). Of note, in OMK cells, cyclosporin A
426 (CsA) was added before the delivery of lentiviral proteins to bypass the Trim-CypA block
427 responsible for capsid destabilization upon entry (36). First, we found that SIVagm Vpr
428 proteins displayed the same phenotypes in OMK cells as in VERO and J-Lat A1 cells in
429 presence of CsA (Fig. 10A: incorporation, Fig. 10B: degradation in OMK cells). Therefore,
430 we extended our panel of lentiviral proteins by testing Vpx proteins from the HIV-2/SIVsmm
431 lineage (HIV-2 Ghana-1 strain and SIVsmm Vpx). As previously shown by us and others (27,
432 28, 37), HIV-2 and SIVsmm Vpx induced human TASOR degradation (Fig. 10C); in
433 addition, we show here that these viral proteins present the same phenotypes in AGM Vervet
434 cells (VERO) (Fig. 10D). Moreover, although SIVagm.Ver, SIVagm.Tan and SIVagm.Sab
435 Vpr proteins had similar phenotypes in owl monkey, AGM and human cells, Vpx proteins
436 from HIV-2 and SIVsmm were unable to induce TASOR degradation in owl monkey cells as
437 opposed to in human and AGM Vervet cells (Fig. 10E). To further confirm TASOR
438 degradation phenotypes in owl monkey cells, we incorporated SIVagm.Ver Vpr and HIV-2
439 Vpx in SIV-derived GFP encoding viruses and found that GFP expression in owl monkey
440 cells was similar (Fig. 10F). This ruled out the hypothesis that Vpx delivery was impaired in
441 owl monkey cells, though we cannot exclude the possibility that the stability of the two viral
442 proteins may differ.

443 Altogether, on top of lentivirus-lineage specificity, we now have one evidence of host-species
444 specificity in the interplay between the HUSH complex and the lentiviral Vpr/Vpx proteins
445 (summary Fig. 10G).

446 Next, we set out to determine whether the apparent host-species specificity could
447 result from a defect of binding of Vpx to DCAF1 and/or TASOR. First, both SIVagm.Ver Vpr
448 and HIV-2 Vpx could interact with owl monkey DCAF1 (Fig. 11A). Unfortunately,
449 interaction of viral proteins with endogenous TASOR is difficult to detect. Thus, we
450 generated by gene synthesis a vector encoding a flag-tagged owl monkey TASOR. The
451 TASOR sequence of the *Aotus trivirgatus* species could not be obtained, we used the
452 publicly-available sequence from a closely related owl monkey species, *Aotus nancymaae*
453 (aotNan; XM_012460781.1). We conducted experiments involving the simultaneous
454 exogenous expression of Flag-tagged owl monkey (aotNan) or human TASOR with, or
455 without, the HA-tagged HIV-2 Vpx or SIVagm.Ver Vpr. By an anti-Flag co-
456 immunoprecipitation assay, we found that both HIV-2 Vpx and SIVagm.Ver Vpr exhibited
457 interaction with human Flag-TASOR but also with owl monkey (aotNan) Flag-TASOR (Fig.
458 11B). These findings suggest that HIV-2 Vpx is able to interact with owl monkey DCAF1 and
459 (aotNan) TASOR despite the lack of degradation of TASOR in owl monkey (aotTri) cells.

460

461 DISCUSSION

462 In the molecular arms-race between pathogenic viruses and their hosts, proteins are submitted
463 to strong selective pressures. Adaptive mutations in the host immune defense may result from
464 the escape from a viral antagonist, while adaptive changes in viral proteins may maintain the
465 virus' ability to counteract the host defense. Taking advantage of natural variations in
466 lentiviruses and host immune defenses from African green monkeys at the inter- and intra-
467 species levels, we show that closely related Vpr proteins can induce the degradation of
468 SAMHD1 variants (haplotypes IV and V) through distinct molecular determinants and that
469 the SIVagm.Ver Vpr protein uses also distinct determinants to trigger SAMHD1 and TASOR
470 degradation. Lastly, we discovered that HIV-2 Vpx proteins from people living with HIV
471 present distinct TASOR degradation abilities, which do not correlate with viremia. TASOR-
472 antagonism determinants are context dependent and are both in the N and C termini. Overall,
473 our results underline the high plasticity of Vpr and Vpx proteins to hijack ubiquitin ligase
474 complexes and to eliminate restrictive host proteins. Lastly, we have evidence of host species-
475 specificity in HUSH antagonism with HIV-2 and SIVsmm Vpx proteins able to induce
476 TASOR degradation in human and AGM VERO cells, but not in owl monkey OMK cells.

477 Only four amino acid differences specify the distinct behavior of the SAMHD1 haplotypes IV
478 and V, with respect to SIVagm Vpr proteins (26). We found that SIVagm.Ver and
479 SIVagm.Tan Vpr both use their C-ter tails to target SAMHD1 haplotype IV. Interestingly, in
480 the crystal structures of the ternary complexes of DCAF1, SAMHD1 and Vpx from SIVsmm,
481 which also targets the C-ter domain of SAMHD1, no residue of the C-terminal tail (also VR3)
482 of Vpx were found in contact with SAMHD1 (23, 24). Nonetheless, such structural studies
483 might not be adapted to Vpx C-ter tail, due to its flexible nature. It is also possible that the
484 recognition mechanism has diverged within the SIVagm lineage or that the C-terminal tail has
485 only a structural role in maintaining the recognition interface.

486 Our approach using chimeric proteins did not allow to reveal specific viral determinants of
487 SIVagm.Gri Vpr involved in haplotype V degradation, suggesting that the integrity of
488 different parts of the viral protein is required. The use of different interfaces by closely related
489 viral proteins underlines how antagonism of a given restriction factor results from different
490 modes of adaptation by the virus. Our results showing that the substitution of only one amino-
491 acid within a given Vpr protein restores its ability to induce the degradation of SAMHD1
492 further supports this model of molecular adaptation along evolution.

493

494 While the C-terminal tail of SIVagm.Ver Vpr conferred on the SIVagm.Gri protein the ability
495 to induce SAMHD1 haplotype IV degradation, the α-helix 3 of SIVagm.Ver Vpr conferred on
496 the SIVagm.Gri protein the ability to degrade the HUSH core protein TASOR. These results
497 suggest that the C-terminal tail and α-helix 3 are key determinants for SAMHD1 haplotype
498 IV and TASOR degradation, respectively, but they do not exclude the possibility that the
499 integrity of other determinants within the viral protein is required for the degradation of each
500 substrate. Indeed, as the SIVagm.Ver and SIVagm.Gri proteins are very similar in sequence,
501 chimera may share similar determinants important for the viral protein activity. In any event,
the use of distinct determinants for the degradation of two different substrates fits with a model of ubiquitin ligase hijacking, in which a viral protein would induce the degradation of different host factors using distinct viral interfaces for substrate recognition, but hijacking only one type of ubiquitin ligase. Intriguingly, SIVagm.Gri Vpr could not induce HUSH degradation, while the integrity of the whole protein seemed required for SAMHD1 haplotype V degradation. Therefore, the selective pressure imposed on the “entire” virus protein to counteract SAMHD1 may have limited its ability to adapt and counteract HUSH (27). Alternatively, it is possible that SIVagm.Gri Vpr cannot degrade human, Vervet AGM or owl monkey TASORs, but can degrade the TASOR from Grivet AGMs, in a host-species specific manner. To address this question, all three components of HUSH (TASOR, MPP8 and periphilin) should be analyzed for polymorphisms and variants within and between AGM species and more extensively in primates, and tested for their degradation in the presence of the different SIVagm Vpr proteins. It is also possible that important cofactors are lacking/different in the cells from other hosts. Altogether, the use of different viral interfaces between closely related Vpr proteins within the SIVagm lineage highlights the dynamism and constraints of the molecular interactions between Vpr proteins, SAMHD1 and HUSH, as a result of a cat-and-mouse game during evolution.

We further highlighted that Vpx proteins of HIV-2 from PLWH-2 harbor different phenotypes. The ability to induce TASOR degradation can be restored after substitution of residues either in the N or in the C-ter part (including helix 3) of the viral protein depending on the Vpx, suggesting that different regions in Vpx could contribute to its interaction with HUSH.

Our study also opens questions concerning the role of HIV-2 Vpx. By inducing HUSH degradation, Vpx is able to increase the proviral transcription from the HIV LTR and the stability of the LTR-driven transcript, thus Vpx counteracts the silencing of the provirus (38). One could think at first that Vpx confers an advantage in HIV-2 replication by enhancing LTR-driven RNA expression. However, interestingly, this study of Vpx proteins derived from both viremic (NCs) or long-term aviremic (EV) PLWH-2 showed that the capacity to induce TASOR degradation did not correlate with the viremia. The importance of TASOR degradation over the course of HIV-2 infections remains to be investigated. In addition, we cannot exclude that some HIV-2 strains may use other non-Vpx determinants in TASOR antagonism.

Lastly, we determined here that HUSH antagonism presents some features of host-species specificity in that the HIV-2/SIVsmm Vpx proteins could induce the degradation of human
and AGM vervet, but not of owl monkey TASOR. The underlying causes remain unknown. It is unlikely a defect in the viral protein delivery, because other Vpr/Vpx proteins were well delivered and functional in these cells, but we cannot exclude that HIV-2/SIVsmm Vpx may be particularly unstable in OMK cells. Our coIP results suggest that absence of degradation does not result from a lack of interaction with DCAF1 or TASOR (35). However, one limitation in our coIP experiments is that, due to challenges in sequencing the entire OMK TASOR, we here tested TASOR from a publicly-available sequence of *Aotus nancymaae* TASOR, which could be slightly different from that of OMK cells (*Aotus trivirgatus*).

Virus-host species-specificity is a hallmark of restriction factors. Antagonism of APOBEC3G by Vif occurs in a species-specific manner in Catharine primates and AGM populations, and functional evolutionary studies showed that the specificity of Vif reflects adaptation of the virus to the host including in key cross-species transmission events (3, 39-46). Experimental virus-host heterologous *in vivo* infection of AGMs showed that the lentiviral *vif* gene can adapt to Vif-resistant APOBEC3G haplotypes (43). Although not observed in the same experimental setting with Vpr (26), SIVagm Vpr proteins have acquired distinct interfaces to counteract SAMHD1, suggesting some adaptation of the virus to SAMHD1 selective pressure. The resistance of owl monkey TASOR to HIV-2/SIVsmm Vpx further suggests that HUSH may have evolved in some primate ancestors in response to past lentiviral epidemics. Alternatively, another pathogen, endogenous viral elements or drivers may have shaped primate TASOR resulting in some host species-specificity. More primate sequences in certain lineages and evolutionary analyses combined with functional assays would help to determine the modes and causes of such host evolution. Altogether, on one hand, SAMHD1 and HUSH antagonisms by Vpr/Vpx proteins appear to be critical components of primate lentiviral fitness witnessed by the complex dynamism of the interactions at stake. On the other hand, results from this study and others (30) suggest that Vpx-mediated SAMHD1 or HUSH antagonism does not always correlate with viral control in PLWH-2. Future work will help to resolve this apparent paradox.

## ACKNOWLEDGMENTS

We thank all the members of the “Retrovirus, Infection and Latency team” for fruitful discussions. We also thank the members of the LP2L team at CIRI, Lyon, for support. We acknowledge the CYBIO, IMAG’IC core facilities of the Institut Cochin.
569 This work was supported by grants from the SIDACTION, the French Research Agency on
570 HIV and Emerging Infectious Diseases ANRS/MIE and Fondation pour la Recherche
571 Médicale (FRM, EQU202203014684 attributed to F.MG.). P.L. was supported by Université
de Paris Cité, M.M.M. by SIDACTION, C.G. by FRM (EQU202203014684 attributed to
572 F.MG.), K.Z. by ANRS/MIE, R.M. by SIDACTION and FRM (EQU202203014684
573 attributed to F.MG.). The work in the laboratory of L.E. is supported by grants from the
574 ANRS/MIE (#ECTZ118944 to LE) and SIDACTION (n°21-1-AEQ-12972-2 to LE and
575 FMG).
576

577

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715 **Figure legends**
716

716

717 **Figure 1: Closely related Vpr proteins, which differently antagonize SAMHD1 variants,**
718 bind DCAF1

719 (A) Schematic representation of SAMHD1 haplotypes III, IV and V. The phenotype of their
720 degradation as shown in B is indicated on the right (v: degradation, x: no degradation). (B)
721 293T cells stably expressing HA-tagged SAMHD1 haplotypes III, IV and V were transfected
722 with plasmids encoding SIVagm Vpr proteins (ver9063: strain of SIVagm.Ver, grv677: strain
723 of SIVagm.Gri). (Left) The indicated proteins were revealed by western-blot (one
724 representative western-blot is shown). (Right) Quantification of SAMHD1 degradation rate
725 from 3 experiments. A one-sample t-test was performed. Data are presented as mean ±
726 standard deviation (SD). Statistical significance is indicated as follows: *p < 0.05, **p < 0.01,
727 ***p < 0.001. (C) HA-Vpr proteins were co-expressed in HeLa cells cultured on glass slides.
728 Cells were fixed and stained with fluorescent probes for DNA (DAPI; blue), HA-Vpr (anti-
729 HA Alexa Fluor 488-conjugated; green). (Left) Images were acquired using confocal
730 microscopy, representative images showing the distribution of HA-Vpr are shown. (Right)
731 Quantification of the mean fluorescence intensity per cell (n = 30 cells) using ImageJ. Error
732 bars represent the standard deviation. No statistically significant difference in fluorescence
733 intensity was observed between SIVagm.Ver, and SIVagm.Gri Vpr (p > 0.05, Student's t-
734 test). (D) HeLa cells were transfected with plasmids encoding SIVagm Vpr proteins. Cells
735 were fractionated before the lysis of the nucleus and the cytoplasm. The indicated proteins
736 were revealed by western blot. (E) Schematic representation of SIVagm Vpr proteins and
737 chimeras. The scale is respected for the length of the different domains. (F) Indicated HA-Vpr
738 constructs were expressed in HeLa cells, then an anti-HA immunoprecipitation was
739 performed, and proteins were revealed by western-blot.

740

741 Figure 2: Closely related Vpr proteins can induce the degradation of SAMHD1 variants
742 (Haplotypes IV and V) through distinct molecular determinants

743 (A) 293T cells stably expressing HA-tagged SAMHD1 haplotype IV were transfected with
744 plasmids encoding SIVagm Vpr proteins and chimeras. (Top) A representative western blot
745 from 3 experiments is shown, (bottom) Quantification of SAMHD1 degradation rate from 3
746 experiments. A one-sample t-test was performed. Data are presented as mean ± standard
747 deviation (SD). Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p <
748 0.001. (B) Same as in (A) but with different Vpr constructs as indicated. (C) Same as in (A)
749 but with cells stably expressing SAMHD1 haplotype V.

750

751 Figure 3: SIV strains of African green monkey species differently degrade TASOR both
752 in Vero and human cells

753 (A) VLPs were produced in 293FT by co-transfection of a packaging vector, an envelope
754 VSVg vector and a vector encoding HA- or Flag-Vpr as indicated. 48h post transfection,
755 supernatants were harvested and VLPs, concentrated by ultracentrifugation, were analyzed by
756 western blot. VLP production was checked with anti-P27 (capsid) antibody and HA-Vpr
757 incorporation with an anti-HA antibody. (B) (Left) VERO cells were treated with VLPs
758 containing SIVagm Vpr proteins as indicated and whole-cell extracts analyzed by western
759 blot. (Middle) Quantification of TASOR degradation rate from 3 experiments. A one-sample
760 t-test was performed. Data are presented as mean ± standard deviation (SD). Statistical
761 significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001. (Right) VERO cells
762 were treated with either siRNA CTRL or siRNA TASOR. (C) and (D) Human J-Lat A1 T
763 cells were treated with VLPs containing SIVagm Vpr proteins and stimulated overnight with
764 TNF-α, then cells were analyzed by western-blot and flow cytometry for the percentage of
765 GFP-positive cells. (C) (Left) Whole-cell extracts were analyzed by western blot, the
766 immunoblot shown is representative of at least 3 independent VLP productions. (Right)
767 Quantification of TASOR degradation rate from 3 experiments. A one-sample t-test was
768 performed. Data are presented as mean ± standard deviation (SD). Statistical significance is
769 indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001. (D) The reactivation rate
corresponds to the percentage of GFP-positive cells in the presence of one viral protein over the percentage obtained without viral protein (empty condition). Reactivation rates from 3 independent experiments are shown, along with raw reactivation data from one representative experiment. A one-sample t-test was performed, with statistical significance denoted as: \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001.

Figure 4: α-helix 3 of SIVagm.Vpr confers on the SIVagm.Gri protein the ability to induce the degradation of the HUSH core protein TASOR

(A) Incorporation of Vpr chimera proteins into VLPs as described in Figure 3A. (B) VERO cells were treated with VLPs containing SIVagm Vpr proteins. After overnight treatment cells were lysed. (Top) Whole-cell extracts were analyzed by western blot, the immunoblot shown is representative of at least 3 independent VLP productions. (Bottom) Quantification of TASOR degradation rate from 3 experiments. A one-sample t-test was performed. Data are presented as mean ± standard deviation (SD). Statistical significance is indicated as follows: \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001. (C) Human J-Lat A1 T cells were treated with VLPs containing SIVagm Vpr proteins. After overnight treatment with TNF-α, cells were analyzed by flow cytometry for the percentage of GFP-positive cells. (Top) Whole-cell extracts were analyzed by western blot, the immunoblot shown is representative of at least 3 independent VLP productions. (Bottom) Quantification of TASOR degradation rate from 3 experiments. A one-sample t-test was performed. Data are presented as mean ± standard deviation (SD). Statistical significance is indicated as follows: \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001. (D) The reactivation rate corresponds to the percentage of GFP-positive cells in the presence of one viral protein over the percentage obtained without viral protein (empty condition). Reactivation rates from 3 independent experiments are shown (left), along with raw reactivation data from one representative experiment (right). A one-sample t-test was performed, with statistical significance denoted as: \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001.

Figure 5: The substitution of only one amino-acid in SIVagm.Gri Vpr restores its ability to induce the degradation of SAMHD1

(A) The C-terminal domain sequences from SIVagm.Ver9063, SIVagm.Grv677, SIVagm.Grv677\* (original sequence) and SIVagm.Tan Vpr proteins were aligned to point out amino acid differences. Substitutions tested in SIVagm.Ver Vpr to assess a functional loss are highlighted in grey. The key residue identified for haplotype IV SAMHD1 degradation is shown in red. (B) 293T cells were transfected with plasmids coding for HA-tagged Vpr
proteins, chimera 9 or mutants, as indicated; (top) extracts from these cells were analyzed by western blot, one representative experiment is shown. (bottom) Quantification of SAMHD1 degradation rate from 3 experiments. A one-sample t-test was performed. Data are presented as mean ± standard deviation (SD). Statistical significance is indicated as follows: \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001. (C) SIVagm.Ver Vpr, SIVagm.Gri Vpr and SIVagm.Gri Vpr G102R were overexpressed by transfection in 293T cells stably expressing the haplotype III or IV of HA-tagged SAMHD1. (top) Extracts from these cells were analyzed by western blot, one representative experiment is shown. (bottom) Quantification of SAMHD1 degradation rate from 3 experiments. A one-sample t-test was performed. Data are presented as mean ± standard deviation (SD). Statistical significance is indicated as follows: \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001. (D) Schematic representation of key viral determinants involved in SAMHD1 and TASOR degradation.

Figure 6: The whole integrity of SIVagm.Ver Vpr α-helix 3 contributes to the degradation of TASOR

(A) α-helix 3 sequences from SIVagm.Ver9063, SIVagm.Grv677, SIVagm.Grv677\* (original sequence) and SIVagm.Tan Vpr proteins were aligned to point out amino acid differences. Substitutions tested in SIVagm.Ver to assess a functional loss are highlighted in grey. Key residues identified for TASOR degradation are shown in red. (B) Same as Fig. 3A with SIVagm.Ver9063 mutants. (C) Human J-Lat A1 T cells were treated with VLPs containing Vpr AGM proteins. After overnight treatment with TNF-α, (top) whole-cell extracts were analyzed by western blot (C) and cells were analyzed by flow cytometry for the percentage of GFP-positive cells (D). (C) The immunoblot shown is representative of at least 3 independent VLP productions. (bottom) Quantification of TASOR degradation rate from 3 experiments. A one-sample t-test was performed. Data are presented as mean ± standard deviation (SD). Statistical significance is indicated as follows: \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001. (D) The reactivation rate corresponds to the percentage of GFP-positive cells in the presence of one viral protein over the percentage obtained without viral protein (empty condition). Reactivation rates from 3 independent experiments are shown (left), along with raw reactivation data from a representative experiment (right). A one-sample t-test was performed, with statistical significance denoted as: \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001. (E) Indicated HA-Vpr constructs were expressed in HeLa cells, then an anti-HA immunoprecipitation was performed, and proteins were revealed by western blot.
838 Figure 7: **TASOR degradation by Vpx proteins from different origins, including HIV-2**
839 **from PLWH-2**

840 Human J-Lat A1 T cells were treated with VLPs containing Vpx proteins from different
841 origins (control Vpx from Ghana-1, Rod and JK strains and four HIV-2 Vpx alleles derived
842 from PLWH-2, according to Yu *et al.*.) and overnight with TNF-α. Whole-cell extracts and
843 Vpx incorporation into VLPs were analyzed by western blot (A and B) and cells were also
844 analyzed by flow cytometry for the percentage of GFP-positive cells (C). (A) (Top) the
845 immunoblot shown is representative of at least 3 independent experiments; (bottom)
846 Quantification of TASOR degradation rate from 3 experiments. A one-sample t-test was
847 performed. Data are presented as mean ± standard deviation (SD). Statistical significance is
848 indicated as follows: *p* < 0.05, **p** < 0.01, ***p** < 0.001. (B) Incorporation of viral proteins
849 into the VLPs used in 9A is checked by western blot. (C) The reactivation rate corresponds to
850 the percentage of GFP-positive cells in the presence of one viral protein over the percentage
851 obtained without viral protein (empty condition). Reactivation rates from 3 independent
852 experiments are shown (left), along with raw reactivation data from one representative
853 experiment (right).

854

855 Figure 8: **The N-ter region of Vpx RH2-1D8 confers on Vpx RH2-7B3 the ability to**
856 **induce the degradation of the HUSH core protein TASOR**

857 (A) Sequences from Vpx RH2-1D8 and Vpx RH2-7B3 were aligned to point out amino acid
858 differences (in red). Cross-referenced sequences for chimera are highlighted in light gray. (B)
859 Representation of the two indicated Vpx proteins and chimera. (C, D, E) Vpx incorporation
860 into VLPs, TASOR degradation in J-Lat A1 T cells following Vpx delivery by VLPs and
861 HIV-1 reactivation were assessed as in Figure 7.

862

863 Figure 9: **The C-ter region of Vpx RH2-1D8 confers on Vpx RH2-22-1B4 the ability to**
864 **induce the degradation of the HUSH core protein TASOR**

865 (A) Sequences from Vpx RH2-1D8 and Vpx RH2-22-1B4 were aligned to point out amino
866 acid differences (in red). The cross-referenced sequence for chimera is highlighted in light
867 gray. (B) Representation of the two indicated Vpx proteins and chimera. (C, D, E) Vpx
868 incorporation into VLPs, TASOR degradation in J-Lat A1 T cells following Vpx delivery by
869 VLPs and HIV-1 reactivation were assessed as in Figure 7.

870
871 Figure 10: **HIV-2/SIVsmm Vpx induces TASOR degradation in human and VERO cells,**
872 **but not in Owl monkey cells**

873 (A) Incorporation of Vpr and Vpx proteins into VLPs as described in Figure 3A. (B) OMK
874 cells were treated with VLPs containing the indicated Vpr/Vpx proteins. After overnight
875 treatment with cyclosporine CsA (or DMSO in control samples), whole-cell extracts were
876 analyzed by western blot. (C) Human J-Lat A1 T cells were treated with VLPs containing
877 Vpx proteins. After overnight treatment with TNF-α, whole-cell extracts were analyzed by
878 western blot, the immunoblot shown is representative of at least 3 independent VLP
879 productions. (D) Same as (C), but in VERO cells. (E) same as B, but with a different set of
880 viral proteins. (F) (Left) SIVagm.Ver Vpr or HIV-2 Vpx were incorporated into SIV-derived
881 viruses that express GFP following cell transduction. Vpx or Vpr incorporation was checked
882 by western blot on the VLPs. (Right) OMK cells were treated with the Vpx or Vpr-containing
883 SIV-derived viruses. After overnight treatment with CsA, whole-cell extracts were analyzed
884 by western blot. (G) Summary of the degradation phenotypes of the lentiviral proteins. Blue:
885 efficient degradation of TASOR and gray: no degradation.

886

887 Figure 11: **HIV-2 Vpx interacts with TASOR and DCAF1 in Owl monkey cells**

888 (A) Indicated HA-Vpr/Vpx constructs were expressed in OMK cells, then an anti-HA
889 immunoprecipitation was performed, and proteins were revealed by western-blot. (B)
890 SIVagm.Ver9063 Vpr or HIV-2.Gh1 Vpx were overexpressed in OMK cells with human or
891 owl Flag-TASOR, then an anti-Flag immunoprecipitation was performed, and proteins were
892 revealed by western-blot.
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

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<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_11.png" /></figure>

https://doi.org/10.1101/2024.03.07.583867

This version is posted February 11, 2025. The copyright holder for this preprint (which may not be certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint (which may not be certified by peer review) is the author/funder, All rights reserved. No reuse allowed without permission.
A

Incorporation into VLPs

<figure><img src="image_12.png" /></figure>

C

J-lat A1 cells

<figure><img src="image_13.png" /></figure>

B

VERO cells

<figure><img src="image_14.png" /></figure>

* aspecific

<figure><img src="image_15.png" /></figure>

D

<figure><img src="image_16.png" /></figure>

Reactivation rate in J-lat A1 cells

(n=3)

<figure><img src="image_17.png" /></figure>

Raw reactivation data for one sample

(n=1)

<figure><img src="image_18.png" /></figure>

bioRxiv preprint doi: https://doi.org/10.1101/2024.07.386138; this version posted February 11, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, All rights reserved. No reuse allowed without permission.
A

<figure><img src="image_19.png" /></figure>

C

293T cells

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

B

293T cells

<figure><img src="image_22.png" /></figure>

D

<figure><img src="image_23.png" /></figure>

bioRxiv preprint doi: https://doi.org/10.1101/2024.03.07.583867; this version posted February 11, 2025. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
<figure><img src="image_24.png" /></figure>

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<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /></figure>
A J-Lat A1 cells (Homoïds)

<figure><img src="image_30.png" /></figure>

B Incorporation into VLPs

<figure><img src="image_31.png" /></figure>

C Reactivation rate in J-lat A1 cells (n=3)

<figure><img src="image_32.png" /></figure>

Raw reactivation data for one sample (n=1)

<figure><img src="image_33.png" /></figure>
<figure><img src="image_34.png" /></figure>

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<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>

bioRxiv preprint doi: https://doi.org/10.1101/2024.03.07.583867; this version posted February 1, 2025. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
<figure><img src="image_39.png" /></figure>

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<figure><img src="image_45.png" /></figure>

bioRxiv preprint doi: https://doi.org/10.1101/2024.03.07.583867; this version posted February 1, 2025. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
<figure><img src="image_46.png" /><figcaption>**A** Incorporation into VLPs</figcaption></figure>

<figure><img src="image_47.png" /><figcaption>**B** OMK cells (NWM)</figcaption></figure>

<figure><img src="image_48.png" /><figcaption>**C** J-Lat A1 cells (Hominoïds)</figcaption></figure>

<figure><img src="image_49.png" /><figcaption>**D** VERO cells (OWM)</figcaption></figure>

<figure><img src="image_50.png" /><figcaption>**E** OMK cells (NWM)</figcaption></figure>

<figure><img src="image_51.png" /><figcaption>**F** Incorporation into VLPs</figcaption></figure>

<figure><img src="image_52.png" /><figcaption>**G** OMK cells (NWM)</figcaption></figure>

<figure><img src="image_53.png" /></figure>

<figure><img src="image_54.png" /></figure>
A OMK cells (NWM)

bioRxiv preprint doi: https://doi.org/10.1101/2024.03.07.583867; this version posted February 11, 2025. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

<figure><img src="image_55.png" /></figure>

B 293T cells

<figure><img src="image_56.png" /></figure>
**Vpr SIVagm.ver9063 (GenBank: KF741096.1):**

MASGRDPRENRPGEVEIWDLNREPWDEWL RDMLEDLNQEAKLHF GRELLFQVWNYCQEEGERRGAPM TERAYRYYRLVQKALFVHFRCGCRRRQPFEPYEERRDGQGGGRANRAPPGLD

**Vpr SIVagm.GRV677 (same as in Spragg and Emerman 2013):**

MTSGRPREPLPGWLEIWDLDREPWDEWLQDMLRDLNEEARRHFGMNMLIRVWNYCVEEGRRHNTPWNEIGYKYYRIVQKSMFVHFRCGCRRRGPFSPYEEGRNGQGGGAPPPPPGLA

**Vpr SIVagm.GRV677* (NCBI Reference Sequence: NP_054371.1):**

MASGRDPREPLPGWLEIWDLDREPWDEWLQDMLRDLNEEARRHFGMNMLIRVWNYCVEEGRRHNTPWNEIGYKYYRIVQKSMFVHFRCGCRRRGPFSPYEEERRNGQGGGAPPPPPGLA

**Vpr SIVagm.SAB1 (PIR: S46351):**

MASGGWPVGGDPPKDPPKNREEIPGWLETWDLPREPFDEWLRDMLQDLNSEA QCHFPRNLLFRLWWNIVEEPAIDHGQTRLEGWYKYCRILQKALFVHMKGRCCPKKTHPAYG PAGGGPPGLGGASGGAA

AAPGL

**Vpr SIVagm.Tan1 (GenBank: U58991.1):**

MAEGDRERRPGWLEIWDLSREPWDEWL RDMVAELNQEAQRHFGRELLFQVWNFCQEGERNGAPMIERAYRYYRLVQKALFVHFRCGCRRRTPFEPYEERRNGVGGGRDGREPPGLA

**Chimeraz1:** MTSGRDPREPLPGWLEIWDLDREPWDEWLQDMLRDLNEEARRHFGMNMLIRVWNYCVEEGRRHNTPM TERAYRYYRLVQKALFVHFRCGCRRRQPFEPYEERRDGQGGGRANRAPPGLD

**Chimeraz2:** MASGRGPRENRPGEVEIWDLNREPWDEWLRDMLEDLNQEAKLHF GRELLFQVWNYCQEEGERRGAPWNEIGYKYYRIVQKSMFVHFRCGCRRRGPFSPYEEGRNGQGGGAPPPPPGLA

**Chimeraz3:** MASGRGPRENRPGEVEIWDLNREPWDEWLRDMLEDLNQEAKLHF GMNMLIRVWNYCVEEGRRHNTPWNEIGYKYYRIVQKSMFVHFRCGCRRRGPFSPYEEGRNGQGGGAPPPPPGLA

**Chimeraz4:** MTSGRDPREPLPGWLEIWDLDREPWDEWLQDMLRDLNEEARRHFGRELLFQVWNYCQEEGERRGAPM TERAYRYYRLVQKALFVHFRCGCRRRQPFEPYEERRDGQGGGRANRAPPGLD

**Chimeraz5:** MASGRGPRENRPGEVEIWDLNREPWDEWLRDMLEDLNQEAKLHF GMNMLIRVWNYCVEEGRRHNTPM TERAYRYYRLVQKALFVHFRCGCRRRQPFEPYEERRDGQGGGRANRAPPGLD

**Chimeraz6:** MTSGRDPREPLPGWLEIWDLDREPWDEWLQDMLRDLNEEARRHFGRELLFQVWNYCQEEGERRGAPWNEIGYKYYRIVQKSMFVHFRCGCRRRGPFSPYEEGRNGQGGGAPPPPPGLA

**Chimeraz7:** MASGRGPRENRPGEVEIWDLNREPWDEWLRDMLEDLNQEARRHFGMNMLIRVWNYCVEEGRRHNTPWNEIGYKYYRIVQKSMFVHFRCGCRRRQPFEPYEERRDGQGGGRANRAPPGLD

**Chimeraz8:** MTSGRDPREPLPGWLEIWDLDREPWDEWLQDMLRDLNEEARRHFGMNMLIRVWNYCVEEGRRHNTPM TERAYRYYRLVQKALFVHFRCGCRRRGPFSPYEEGRNGQGGGAPPPPPGLA

**Chimeraz9:** MTSGRDPREPLPGWLEIWDLDREPWDEWLQDMLRDLNEEARRHFGMNMLIRVWNYCVEEGRRHNTPWNEIGYKYYRIVQKSMFVHFRCGCRRRQPFEPYEERRDGQGGGRANRAPPGLD

**Vpx HIV-2:** UniProtKB/Swiss-Prot: P18045.1

MTDPRETPPPGNSGEETIGEAFAWLDRTIEALNREAVNHLPRELIFQVWQRSWRYWHDDQGMSPSYTKYRYLCLMQKAVFIHFKGCTCLGGGHGPGGWRSGPPPPPPGLV

**Vpx HIV-2:** J.K (GenBank: AAL31353.1):

MDPRERVPPGNSDEETVGEAFAWLERTIVELNREAVNHLPRELIFQVWQRSWAYWRDDQGMSISYTKYRYLLLIQKAMFVHFAKGCTCLRGGHGPGGWRGRPPPPPPGLA

**Vpx HIV-2:** A.Rod (UniProtKB/Swiss-Prot: P06939.1):

MTDPRETPPPGNSGEETIGEAFAWLRTVEAINREAVNHLPRELIFQVWQRSWRYWHDEQGMSESYTKYRYLCIIQKAVYMHVRKGCTCLGRGHGPGGWRPGPPPPPPGLV

**Vpx HIV-2:** RH2-13 5C1 (Effective controller):

MADPREAVPPGNSGEETIGEAFAWLERTVEAINREAVNHLPRELIFQVWQKSWRYWHDDQGMSQSYTKYRYLCLIQKALFIHAKKGCTCLGEGH GPGGWRSGPPPPPPGLV

**Vpx HIV-2:** RH2-22-1B4 (Effective controller):

MDPRERVPPGNSGEETVGEAFAWLERTVEAINREAVNHLPRELIFQVWQRSWRYWHDEQGMSSYTKYRYLCLMQKAMYIHFAKGCTCLREGH GPGGWRPGPPPPPPGLA

**Vpx HIV-2:** RH2-1D8 (Non controller):

MADPREIVPPGNSGEETVGEAFAWLERTVEAINREAVNHLPRELIFQVWQRSWRYWHDEQGMSSYTKYRYLCLMQKAMFIHFRGCTCLGGGHGPGGWGPPPPPPPGLV

**Vpx HIV-2:** RH2-7B3 (Non controller):

MVDPRETPPPGNSGEETIGEAFAWENRTVEAINREAVNHLPRELIFQVWQRSWRYWHDEQGMSQSYTKYRYLCIIQKALFIHAKQGCTCRGEGH GPGGWRSGPPPPPPGLV

**Chimeraz8B3:** MADPREIIPPPGNSGEETVGEAFAWLERTVEAINREAVNHLPRELIFQVWQRSWRYWHDEQGMSQSYTKYRYLCIIQKALFIHAKQGCTCRGEGH GPGGWRSGPPPPPPGLV

**Chimeraz8B3:** MVDPRETVPPGNSGEETIGEAFAWENRTVEAINREAVNHLPRELIFQVWQRSWRYWHDEQGMSQSYTKYRYLCLMQKAMFIHFRGCTCLGGGHGPGGWRSGPPPPPPGLV

**Chimeraz8B4:** MDPRERVPPGNSGEETIREAFEWLERTVAELNREVNHLPRELIFQVWQRSWAYWHEEQGMSASYTKYRYLCLMQKAMFIHFRGCTCLGGH GPGGWRPGPPPPPPGLA