Evolutionary and Functional Analyses of the Interaction between the Myeloid Restriction Factor SAMHD1 and the Lentiviral Vpx Protein

Article (PDF Available)inCell host & microbe 11(2):205-17 · February 2012with89 Reads
DOI: 10.1016/j.chom.2012.01.007 · Source: PubMed
Abstract
SAMHD1 has recently been identified as an HIV-1 restriction factor operating in myeloid cells. As a countermeasure, the Vpx accessory protein from HIV-2 and certain lineages of SIV have evolved to antagonize SAMHD1 by inducing its ubiquitin-proteasome-dependent degradation. Here, we show that SAMHD1 experienced strong positive selection episodes during primate evolution that occurred in the Catarrhini ancestral branch prior to the separation between hominoids (gibbons and great apes) and Old World monkeys. The identification of SAMHD1 residues under positive selection led to mapping the Vpx-interaction domain of SAMHD1 to its C-terminal region. Importantly, we found that while SAMHD1 restriction activity toward HIV-1 is evolutionarily maintained, antagonism of SAMHD1 by Vpx is species-specific. The distinct evolutionary signature of SAMHD1 sheds light on the development of its antiviral specificity.
Cell Host & Microbe
Article
Evolutionary and Functional Analyses of the
Interaction between the Myeloid Restriction
Factor SAMHD1 and the Lentiviral Vpx Protein
Nadine Laguette,
1,7
Nadia Rahm,
2,7
Bijan Sobhian,
1
Christine Chable-Bessia,
1
Jan Mu¨ nch,
3
Joke Snoeck,
2,4
Daniel Sauter,
3
William M. Switzer,
5
Walid Heneine,
5
Frank Kirchhoff,
3
Fre
´
de
´
ric Delsuc,
6,
*
Amalio Telenti,
2,
*
and Monsef Benkirane
1,
*
1
Institut de Ge
´
ne
´
tique Humaine, Centre National de la Recherche Scientifique, Unite
´
Propre de Recherche 1142, Laboratoires de Virologie
Mole
´
culaire, 34000 Montpellier, France
2
Institute of Microbiology, University Hospital Center and University of Lausanne, 1011 Lausanne, Switzerland
3
Institute of Molecular Virology, Ulm University Medical Center, 81089 Ulm, Germany
4
Rega Institute for Medical Research, Katholieke Universiteit Leuven, 3000 Leuven, Belgium
5
Laboratory Branch, Division of HIV AIDS Prevention, National Center for HIV, Hepatitis, STD, and TB Prevention, Centers for Disease Control
and Prevention, Atlanta, GA 30333, USA
6
Institut des Sciences de l’Evolution, Unite
´
Mixte de Recherche 5554, Centre National de la Recherche Scientifique, Institut de Recherche
pour le De
´
veloppement, Universite
´
Montpellier II, 34095 Montpellier, France
7
These authors contributed equally to this work
*Correspondence: frederic.delsuc@univ-montp2.fr (F.D.), amalio.telenti@chuv.ch (A.T.), bmonsef@igh.cnrs.fr (M.B.)
DOI 10.1016/j.chom.2012.01.007
SUMMARY
SAMHD1 has recently been identified as an HIV-1
restr iction fact or operating in myeloid cells. As a
coun termeasure, the Vpx accessory protein from
HIV-2 an d certain lineages of SIV have evolved to
antagonize SAMHD1 by inducing its ubiquitin-pro-
teasome-dependent degradation. Here, we show
that SAMHD1 experienced strong positive selection
episodes during primate evolution th at occurred
in the Catarrhini ancestral branch prior to the
separation between hominoids (gibbons and great
apes) and Old World monkeys. The identification
of SAMHD1 residues under positive selection led
to mapping th e Vpx-interaction domain of SAMHD1
to its C-terminal region. Importantly, we found that
while SAMHD1 restriction activity toward HIV-1 is
evolutionaril y maintained, antag onis m of SAMHD1
by Vpx is species-specific. The distinct evolu-
tionary signature of SAMHD1 sheds light on the
development of its antiviral specificity.
INTRODUCTION
Eukaryotic organisms have been exposed to viral infections for
millions of years. This coevolutionary process has driven the
development and adaptation of immune responses against
invading viruses. In turn, viruses have evolved countermeasures
to escape immune control. The human immunodeficiency virus
(HIV) targets susceptible cells of the immune system that
express the CD4 receptor and coreceptors (CCR5 or CXCR4)
necessary for viral entry. However, certain cells of the immune
system are nonpermissive to HIV-1 infection despite efficient
entry. Indeed, dendritic cells (DCs) are largely refractory to
HIV-1 infection (Coleman and Wu, 2009). This block to viral
replication is linked to the presence of the cellular dominant
negative factor SAMHD1 (Laguette et al., 2011). Three other
cellular proteins have previously been shown to restrict HIV
infection: tripartite motif 5 alpha (TRIM5a), APOBEC3G (apolipo-
protein messenger RNA-editing enzyme catalytic polypeptide-
like editing complex 3 [A3G]) and BST-2/tetherin (Neil et al.,
2008; Sheehy et al., 2002; Stremlau et al., 2004). These restric-
tion factors are all induced by interferon (IFN) treatment,
act on discrete steps of the viral replication cycle, and are
ubiquitously expressed in the organism (Douville and Hiscott,
2010). TRIM5a causes untimely uncoating after delivery of the
viral core in the cytoplasm of target cells (Stremlau et al.,
2006). A3G causes hypermutations of the viral genome, thus
rendering it unstable, unable to integrate or to give rise to new
functional viral particles (Harris et al., 2003). BST-2 tethers
viral particles to the cell surface and therefore prevents viral
budding (Neil et al., 2008 ). The precise step upon which
SAMHD1 acts remains to be fully unraveled. Nonetheless,
SAMHD1 silencing in DCs favors accumulation of full-length
HIV-1 DNA, suggesting that it may affect reverse transcription
(Laguette et al., 2011).
SAMHD1 belongs to a family of proteins that have been
involved in a rare genetic disorder, the Aicardi-Goutie
`
res
Syndrome (AGS) (Rice et al., 2009). Sequencing the genome of
AGS patients revealed mutations in TREX1, RNASEH2A-C, and
SAMHD1 (Crow et al., 2006a, 2006b; Crow and Rehwinkel,
2009). TREX1 and RNaseH2 both play important roles in nucleic
acid metabolism (Mazur and Perrino, 1999), suggesting a similar
mode of action for SAMHD1. Notwithstanding the involvement of
these proteins in similar pathways, their impact on HIV replication
is divergent. TREX1 and RNASH2 are both facilitating factors
(Genovesio et al., 2011; Stetson et al., 2008; Yan et al., 2010 ),
Cell Host & Microbe 11, 205–217, February 16, 2012 ª2012 Elsevier Inc. 205
whereas SAMHD1 is a bona fide restriction factor (Hrecka
et al., 2011; Laguette et al., 2011). AGS patients experience
increased leukocytosis and IFN plasma levels (Dale et al., 2010;
Rice et al., 2009). As it is the case for the TRIM family of proteins,
SAMHD1 appears to be at the crossroads of inflammation and
restriction.
Genetic conflict between hosts and lentiviruses leads to
rapid selection of mutations that alter amino acid composition
of both actors, especially at positions involved in protein-
protein interaction (Emerman and Malik, 2010; Ortiz et al.,
2009). This process of positive selection characterizes A3G
interaction with Vif (Sawyer et al., 2004), TRIM5a interaction
with CA (Sawyer et al., 2005), and BST-2 interaction with Vpu,
Nef, or Env (McNatt et al., 2009; Sauter et al., 2009). While
HIV-1 has no means to counteract SAMHD1 restriction, HIV-2
and certain SIV strains encode the auxiliary protein Vpx that
potently overcomes the block to viral replication constituted
0
50
100
150
200
250
300
350
0 0,01 0,02 0,03 0,04 0,05 0,06 0,07 0,08 0,09 0,1
A
B
SAMHD1
C
0.0058 (concatenation)
0.0029 (median)
0.03
substitution / site
Tarsius syrichta
Macaca mulatta
Microcebus murinus
Pongo abelii
Tupaia belangeri
Gorilla gorilla
Callithrix jacchus
Homo sapiens
Otolemur garnettii
Pan troglodytes
0.0058
Callithrix jacchus
Microcebus murinus
Macaca mulatta
Tarsius syrichta
Tupaia belangeri
Pongo abelii
Pan troglodytes
Otolemur garnettii
Homo sapiens
Gorilla gorilla
0.1032
0.09
substitution / site
SAMHD1 CDS
786 orthologous CDS
Platyrrhini (NWM)
Catarrhini (Hom/OWM)
Prosimians
PRIMATES
MAMMALS
Scandents
Platyrrhini (NWM)
Catarrhini (Hom/OWM)
Prosimians
PRIMATES
MAMMALS
Scandents
Figure 1. Characterization of the Atypical
Catarrhini Ancestral Branch Length in the
SAMHD1 Gene Tree
(A) Amino acid branch lengths inferred for
SAMHD1 sequences available for 34 mammals
in the OrthoMaM database (Ranwez et al., 2007)
as inferred by maximum likelihood under the
LG+G+F model implemented in RAxML (Stama-
takis, 2006). Only the Scandentia+primates sub-
tree is presented. Note the extremely long
ancestral branch (0.1032 substitutions per site)
leading to Old World monkeys (OWMs, Catarrhini)
represented in red.
(B) Amino acid branch lengths inferred from a
concatenation of 786 1:1 orthologous CDSs
available for the same 34 taxa in the OrthoMaM
database. Note the much shorter Catarrhini
ancestral branch (0.0058 substitutions per site)
represented in red.
(C) Distribution of the ancestral Catarrhini amino-
acid branch length inferred across the 786 1:1
orthologous gene trees showing that SAMHD1 is
an extreme outlier with a branch length about
17 times longer than the one inferred from the
concatenation.
See also Figure S1 and Table S1.
by SAMHD1 by promoting its degrada-
tion by the proteasome machinery
(Hrecka et al., 2011; Laguette et al.,
2011). Thus, SAMHD1 is expected to be
in a genetic conflict with the lentiviral
protein Vpx.
Here, we show that SAMHD1 experi-
enced strong positive selection episodes
during primate evolution. The identifica-
tion of SAMHD1 residues under positive
selection allowed mapping of the interac-
tion domain with Vpx to the C-terminal
region. Furthermore, we demonstrate
that SAMHD1 proteins of apes, monkeys,
and lemurs are all active against HIV-1,
whereas Vpx degrades and antagonizes SAMHD1 in a
species-specific manner.
RESULTS
SAMHD1 Evolved under Strong Selective Pressures
that Occurred along the Catarrhini Ancestral Branch
To determine whether selection pressure has shaped SAMHD1
evolution, we first constructed a phylogenetic tree using
SAMHD1 coding sequences (CDSs) from 34 different mammals,
including primates, available from ongoing sequencing projects
and present in the database of Orthologous Mammalian Markers
(OrthoMaM) (Ranwez et al., 2007). SAMHD1 showed an atypical
gene tree relative to other 1:1 orthologous genes represented in
the OrthoMaM database. Indeed, the maximum likelihood (ML)
phylogenetic tree inferred from SAMHD1 amino acid sequences
(Figure 1A) exhibited an exceptionally long ancestral branch for
Cell Host & Microbe
Evolution of the SAMHD1/Vpx Interaction
206 Cell Host & Microbe 11, 205–217, February 16, 2012 ª2012 Elsevier Inc.
Catarrhini, including Old World monkeys (OWMs) and homi-
noids. This branch was more than 17 times as long in the
SAMHD1 gene tree (0.1032 substitution/site) as in the ML phylo-
genetic tree (0.0058 substitution/site) inferred from the concate-
nation of the 786 1:1 orthologous CDS for which the same 34
mammalian taxa are available in OrthoMaM (Figure 1B). The
distribution of the ancestral Catarrhini branch length across the
same 786 ML gene trees confirms that SAMHD1 is an extreme
outlier (Figure 1C and Figure S1 available online). The atypical
branch length of the SAMHD1 gene tree underlines an excep-
tional accumulation of amino acid substitutions in this gene,
suggesting the occurrence of an episode of adaptive evolution
in the ancestral lineage leading to Catarrhini.
To test this hypothesis, we sequenced the complete SAMHD1
open reading frame (ORF; approximately 1880 bp) from 25
primate species ( Table S1 and Figure S1). ML analysis of a
32-taxa data set allowed reconstruction of a well-resolved
phylogeny of primates including seven New World Monkeys
(NWMs, Platyrrhini) and 20 OWMs and hominoids (Catarrhini)
(Figure 2A). The inferred phylogeny is almost fully compatible
with the reference topology obtained from the most recent
primate multigene phylogeny (Perelman et al., 2011), except
for some difficulties to resolve phylogenetic relationships
between short internal branches within OWMs and NWMs. Using
the ML topology, we performed a number of statistical tests
based on the ratio of nonsynonymous (dN) to synonymous (dS)
substitutions (dN/dS) to define the selective pressures acting
on the SAMHD1 molecule in primates (Table 1). Estimation of
the global dN/dS ratio across the whole tree using the one-ratio
branch model (M0) resulted in a u value of 0.36, showing that
SAMHD1 is globally under purifying selection in primates.
However, hierarchical likelihood ratio tests (LRTs) performed
between different branch models allowing u to vary among
branches of the phylogeny (Yang, 1998) reveals that strong posi-
tive selection occurred in the Catarrhini ancestral branch
(Table 1). Indeed, the alternative two-ratio model (M2u), that
allows the single ancestral Catarrhini branch to have its own u,
results in a significant increase in likelihood over the one-ratio
M0 model (p < 0.001). Under this two-ratio model, u is estimated
to be 1.27 along the ancestral branch leading to Catarrhini, indi-
cating episodes of positive selection. Overall, the best-fitting
branch model distinguishes three categories for u distributed
across the phylogeny. This model confirms that positive selec-
tion occurred along the Catarrhini ancestral branch (u = 1.30),
followed by a decrease in Old World monkeys and hominoids
(u = 0.59) whereas New World Monkeys and other primates
present a dN/dS ratio characteristic of purifying selection
(u = 0.26) (Figure 2A). Such branch models nevertheless require
a priori choice of the branches or group of branches of the phylo-
genetic tree among which u might differ. To relax this assump-
tion, we used a recently developed alternative approach that
consists in an integrated Bayesian framework that allows joint
reconstruction of variations in the molecular evolutionary
process, including dN/dS ratios, life-history traits and diver-
gence times (Lartillot and Poujol, 2011). We used this approach
to reconstruct the variation of the dN/dS ratio in SAMHD1 along
primate phylogeny while controlling divergence times and the
effect of three life-history traits: body mass, longevity, and
maturity (Figure 2B). The Bayesian reconstruction shows a clear
increase of the dN/dS ratio in the Catarrhini ancestral branch
length (mean dN/dS = 1.05) with elevated values persisting in
Hominidae, especially in orangutans, while Platyrrhini show
uniformly low dN/dS ratios. Moreover, the Bayesian chronogram
infers that positive selection episodes experienced by SAMHD1
during primate evolution occurred approximately 45 to 25 million
years ago, just before diversification of Catarrhini and their
separation into hominoids and cercopithecoids.
These observations lead us to explore SAMHD1 restriction
activity across evolution. For this purpose, we analyzed the
anti-HIV-1 activity of a panel of SAMHD1 orthologs from homi-
noids, OWMs, NWMs, and prosimians (Figure 2C). SAMHD1-
silenced THP-1 myeloid cells or U937 promyeloid cells—that
express no detectable levels of endogenous SAMHD1—were
transduced
with retroviral vectors to express human (hu) short
hairpin RNA (shRNA) resistant SAMHD1 (H1.4R) or a panel of
primate SAMHD1. Cells were differentiated and subsequently
infected with a vesicular stomatitis virus G protein pseudotyped
HIV-1 molecular clone that harbors the luciferase gene in place
of nef as a reporter—HIV-LUC-G. Measuring the luciferase
activity shows that all tested SAMHD1 alleles were able to
restrict HIV-1 infection (Figure 2C and Figure S2). Next, we asked
whether SIV
mac239
is restricted by huSAMHD1. We infected
differentiated THP-1 with either a wild-type (WT) SIV
mac239
molecular clone that possesses an IRES-eGFP sequence as
a reporter or its DVpx counterpart. Infection with SIV
mac239
causes a net decrease of SAMHD1 levels as observed by immu-
nofluorescence, whereas infection with SIV
mac239
DVpx causes
no significant variation in SAMHD1 levels (Figure 2D and Figures
S2D and S2E). Moreover, infection of parental THP-1 cells or
THP-1 cells stably expressing Vpx
mac251
(THP-1-Vpx) with the
same molecular clones of SIV
mac239
shows, in a flow cytometry
assay, that expression of Vpx
mac251
restores SIV
mac239
DVpx
infection to levels similar to those of SIV
mac239
(Figure 2D). This
suggests that optimal SIV
mac239
infection of THP-1 cells requires
the presence of a functional Vpx. Taken together, these experi-
ments show that SAMHD1 restriction activity has been
conserved throughout the evolutionary history of primates.
Identification of Positive Selected Sites Clustered
in SAMHD1 C-Terminal Domain
In order to further characterize episodes of molecular adaptation
that occurred in SAMHD1 and identify putative Vpx-interacting
domains, we explored branch-site models for detection of posi-
tive selection on SAMHD1 domains. These models, that allow u
variation among sites in the protein and across branches on the
tree, aim at detecting positive selection that affects a few sites
along particular lineages (Yang and Nielsen, 2002). The
branch-site tests 1 and 2 (Zhang et al., 2005) were applied to
the Catarrhini ancestral branch, which was set as the foreground
branch. The conservative branch-site test 1 was not significant,
whereas the branch-site test 2 was significant (p < 0.05). Under
branch-site model A, the empirical Bayes procedure did not
identify positively selected sites with significant posterior proba-
bility along the Catarrhini ancestral branch (data not shown).
We investigated the among-site dN/dS ratio variation along
the SAMHD1 molecule using site-specific codon models for
detection of positively selected sites (Nielsen and Yang, 1998;
Yang et al., 2000). Both LRT computed between nearly neutral
Cell Host & Microbe
Evolution of the SAMHD1/Vpx Interaction
Cell Host & Microbe 11, 205–217, February 16, 2012 ª2012 Elsevier Inc. 207
models and models allowing for site-specific positive selection
rejected the null hypothesis of nearly neutral evolution of
SAMHD1 (p < 0.001) with the identification of sites under positive
selection (Table 1). The site-specific selection profile of SAMHD1
inferred under the M8 model revealed 17 positively selected
codons with a posterior probability of more than 0.95 (Figure 3A).
The more conservative but less powerful M2a model identified
four of these codons at positions 256, 408, 601, and 614 to be
under positive selection. One of these codons (position 256) falls
within the functional HD domain of the molecule. Strikingly, we
identified a cluster of positively selected sites in the C-terminal
part of the protein, comprising five sites (positions 601, 602,
A
B
0.04
Homo sapiens*
Allenopithecus nigroviridis*
Alouatta palliata*
Mandrillus sphinx*
Ateles geoffroyi*
Pan troglodytes*
Cercopithecus diana*
Macaca fascicularis*
Miopithecus talapoin*
Cebus apella*
Colobus angolensis*
Pongo pygmaeus*
Hylobates lar*
Nomascus leucogenys
Callithrix jacchus*
Chlorocebus tantalus*
Aotus trivirgatus*
Varecia variegata*
Otolemur garnettii
Saimiri sciureus*
Saguinus oedipus*
Theropithecus gelada*
Pongo abelii
Gorilla gorilla
Macaca mulatta*
Microcebus murinus*
Tupaia belangeri
Trachypithecus francoisi*
Pygathrix nemaeus*
Papio hamadryas*
Cercocebus galeritus*
Tarsius syrichta
= 1.30
= 0.26
NWM
OWM
= 0.59
Prosimians
N
W
M
O
W
M
Paleocene Eocene Oligocene Miocene Pli. P.
065
424355
52
Myr
=1.05
Trachypithecus francoisi*
Hylobates lar*
Tarsius syrichta
Alouatta palliata*
Gorilla gorilla
Cercocebus galeritus*
Macaca mulatta*
Theropithecus gelada*
Mandrillus sphinx*
Pygathrix nemaeus*
Allenopithecus nigroviridis*
Papio hamadryas*
Ateles geoffroyi*
Saguinus oedipus*
Saimiri sciureus*
Nomascus leucogenys
Miopithecus talapoin*
Aotus trivirgatus*
Pongo pygmaeus*
Varecia variegata*
Otolemur garnettii
Cebus apella*
Chlorocebus tantalus*
Microcebus murinus*
Macaca fascicularis*
Homo sapiens*
Callithrix jacchus*
Pan troglodytes*
Tupaia belangeri
Pongo abelii
Colobus angolensis*
Cercopithecus diana*
P
R
O
Hom.
C
Fold increase (luciferase activity)
0
0,2
0,4
0,6
0,8
1
1,2
Hom. OWM NWM
Pro.
SAMHD1:
H
O
M
SAMHD1 DAPI MERGE
SIVmac239SIVmac239Δ Vpx
Fold increase GFP+ cells
D
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
-WT
Δ Vpx
THP-1
THP-1-Vpx
SIVmac239:
ω
1
ω
2
ω
ω
3
Figure 2. Evolutionary Conservation of SAMHD1 Restriction Activity against HIV-1
(A) SAMHD1 phylogenetic tree for 31 primate species rooted by the treeshrew Tupaia belangeri. The topology was inferred by maximum likelihood from the
nucleotide sequences under a GTR+G model using RAxML. This topology is fully congruent with the most recent multigene phylogeny obtained by Perelman et al.
(2011). Branch lengths are those inferred by Codeml under the best fitting branch model (Yang and Nielsen, 1998). Note the episode of positive selection along the
Catarrhini ancestral branch (u = 1.30), followed by a decrease in OWMs and hominids (u = 0.59), whereas New World monkeys (NWMs, Platyrrhini) and other
primates show a dN/dS characteristic of purifying selection (u = 0.26). * indicates sequenced SAMHD1 in this study. Scale is in number of substitutions per codon.
(B) The variation of u in SAMHD1 along the primate phylogeny was jointly reconstructed with divergence times while controlling the effect of three life-history traits
(body mass, longevity, and maturity) using the Bayesian framework recently proposed by Lartillot and Poujol (2011). The Bayesian chronogram obtained shows
that the positive selection episode experienced by SAMHD1 during primate evolution occu rred along the ancestral Catarrihini approximately between 45 and
25 million years ago. Time scale is expressed in million years with vertical lines delimitating the main geological periods (Pli., Pliocene; P., Pleistocene).
(C) SAMHD1 expression was induced through retroviral transduction of SAMHD1-silenced THP-1 myeloid cells. Forty-eight hours after transduction, cells were
differentiated and infected with a HIV-LUC-G. Luciferase activity was measured 24 hr after infection, normalized for protein levels and represented as relative
infection compared to untransduced cells. Mock, untransduced; H1.4R, shRNA-resistant huSAMHD1; Gib, gibbon; Mng, mangabey; Rh, rhesus macaque; Agm,
African green monkey; Tam, tamarin; Owl, owl monkey; Gml, gray mouse lemur; OWM, Old World monkeys; NWM, New World monkeys.
(D) THP-1 and THP-1 stably expressing Vpx were differentiated on coverslips and infected with a SIVmac239-IRES-eGFP molecular clone with an IRES-eGFP
sequence as a reporter (SIVmac239) or with the same virus with a stop codon in the vpx ORF (SIVmac239DVpx). Cells were analyzed by immunofluorescence
48 hr after infection for SAMHD1 expression. Nuclei were stained with DAPI. Cells were further analyzed by flow cytometry for the expression of GFP. Results are
presented as the percent of GFP-positive cells, with the percent of GFP+ THP-1 cells infected with SIVmac239 set to 1. Error bars represent the standard
deviation from the mean.
See also Figure S2.
Cell Host & Microbe
Evolution of the SAMHD1/Vpx Interaction
208 Cell Host & Microbe 11, 205–217, February 16, 2012 ª2012 Elsevier Inc.
614, 618, and 626) that localized within the last 25 amino acids of
SAMHD1 (Figure 3B).
Vpx/SAMHD1 Interaction Is Mediated by the C-Terminal
Domain of SAMHD1
Based on the assumption that genetic conflict between viral
proteins and restriction factors leads to the rapid fixation of
mutations at the site of protein-protein interaction, we hypothe-
sized that the C-terminal domain of SAMHD1 might be involved
in interaction with Vpx. To test this hypothesis, we generated
constructs where human FLAG-tagged wild-type SAMHD1
(WT-SAMHD1) is truncated for either the N-terminal SAM
domain (DSAM) or portions of the C-terminal tail (SAMHD1-
F575, SAMHD1-F595, SAMHD1-F611; Figure 4A). The mutant
SAMHD1-HD/AA, that has no restriction activity toward HIV-1
(Laguette et al., 2011), was used as a control. Like WT-SAMHD1,
all truncated SAMHD1 mutants localized to the nucleus as
demonstrated by immunofluorescence in HeLa cells (Figure S4).
We tested the ability of HA-tagged Vpx from SIV
mac251
(Vpx
mac251
) to degrade D SAM, HD/AA and F575 as compared
to WT-SAMHD1, in 293T cells (Figure 4B). Vpx
mac251
expression
correlated with decreased levels of WT-SAMHD1, SAMHD1-
DSAM, and SAMHD1-HD/AA, whereas no significant decrease
of SAMHD1-F575 levels was observed. We next tested the
ability of Vpx
mac251
to interact with SAMHD1 mutants. FLAG-
tagged WT-SAMHD1, SAMHD1-F575, SAMHD1-F595 were
coexpressed with HA-tagged Vpx
mac251
in 293T cells. FLAG
immunoprecipitation of whole-cell extracts reveals the interac-
tion of Vpx
mac251
with WT-SAMHD1 whereas no interaction
with SAMHD1-F575 and SAMHD1-F595 was detected (Fig-
ure 4C). This indicates that the interaction of SAMHD1 with
Vpx
mac251
is mediated by the last 31 amino acids of SAMHD1
that comprises amino acids 601, 602, 614, 618, and 626, which
are under significant positive selection. To further map this
interaction, we generated a fragment of SAMHD1 (SAMHD1-
F611) that allows discriminating between amino acids 601/602
and 614/618/626. The fact that SAMHD1-F611 fails to interact
with Vpx indicates that the last 11 residues of SAMHD1 (Fig-
ure 4D) comprising amino acids 614, 618, and 626 are involved
in the SAMHD1/Vpx interaction. We next generated point
mutants of SAMHD1 where amino acids S614, V618, and
M626 are substituted by alanines and tested their ability to
interact with Vpx
mac251
. Whole-cell extracts of 293T cells coex-
pressing FLAG-SAMHD1-S614A, -V618A, or -M626A together
with HA-Vpx
mac251
were subjected to FLAG immunoprecipita-
tion. Analysis by immunoblot revealed that SAMHD1-S614A,
SAMHD1-V618A, both interacted with Vpx
mac251
at levels
similar to WT-SAMHD1, whereas SAMHD1-M626A failed to
interact with Vpx
mac251
(Figure 4D). Taken together our results
demonstrate that the C-terminal region of huSAMHD1 is
required for its interaction with Vpx
mac251
and is therefore
critical for Vpx
mac251
-induced degradation of SAMHD1. We
further map amino acid M626 of huSAMHD1 as required for
this interaction.
Vpx-Induced huSAMHD1 Degradation Depends
on Vpx/SAMHD1 Interaction and Involves Relocalization
of SAMHD1 to the Cytoplasm
We previously observed that, when expressed in THP-1
cells, Vpx from SIV
mac251
, and HIV-2
ROD
(Vpx
ROD
) potently
Table 1. Results of Likelihood Ratio Tests for Positive Selection in SAMHD1
Hypotheses LRT
Null Hypothesis Alternative Hypothesis 2DLnL df p Value
Branch Models
M0: one-ratio model M2u: two-ratio model
a
20.46 1 p < 0.001***
M0: one-ratio model M3u: three-ratio model
b
46.92 2 p < 0.001***
M0: one-ratio model M4u: four-ratio model
c
48.76 3 p < 0.001***
M0: one-ratio model M5u: five-ratio model
d
49.13 4 p < 0.001***
M2u: two-ratio model M3u: three-ratio model 26.46 1 p < 0.001***
M2u: two-ratio model M4u: four-ratio model 28.31 2 p < 0.001***
M2u: two-ratio model M5u: five-ratio model 28.67 3 p < 0.001***
M3u: three-ratio model M4u: four-ratio model 1.85 1 ns
M3u: three-ratio model M5u: five-ratio model 2.21 2 ns
M4u: four-ratio model M5u: five-ratio model 0.36 1 ns
Branch-Site Models
M1a (nearly neutral) Model A (u
2
R 1) 3.26 1 ns
Null model A (u
2
= 1) Model A (u
2
R 1) 2.78 1 p < 0.05*
Site Models
M7: beta M8: beta and u 40.41 2 p < 0.001***
M1a: nearly neutral M2a: positive selection 50.92 2 p < 0.001***
***, highly significant; *, significant; ns, not significant.
a
Ancestral Catarrhini branch versus all other branches.
b
Ancestral Catarrhini branch, Catarrhini versus all other branches.
c
Ancestral Catarrhini branch, Catarrhini, Platyrrhini versus all other branches.
d
Ancestral Catarrhini branch, Hominoidea, Cercopithecoidea, Platyrrhini versus all other branches.
Cell Host & Microbe
Evolution of the SAMHD1/Vpx Interaction
Cell Host & Microbe 11, 205–217, February 16, 2012 ª2012 Elsevier Inc. 209
induced huSAMHD1 degradation whereas Vpx from SIV
Rcm-ng
(Vpx
Rcm-ng
) failed to affect huSAMHD1 levels (Laguette et al.,
2011). To extend this observation, we analyzed the ability of
Vpx from additional HIV-2 and SIV strains to cause huSAMHD1
degradation in THP-1 cells, using Vpx
mac251
, Vpx
ROD
, and
Vpx
Rcm-ng
as controls. In the tested panel of Vpx alleles,
we included both alleles from laboratory-adapted lentiviral
strains—Vpx from SIV
mac239
(Vpx
mac239
), Vpx
mac251
, and
Vpx
ROD
—and from ‘primary’ viral strains—SIV sooty mangabey
(Vpx
Smm
), mandrill (Vpx
mnd2
), drill (Vpx
drl1
), HIV-2 A (Vpx
2A
), and
HIV-2 B (Vpx
2B
)(Figure 5). These alleles were all engineered to
be FLAG and HA tagged and inserted in a retroviral vector allow-
ing for transduction of proliferating THP-1 cells. Immunostaining
of SAMHD1 and Vpx showed no significant costaining of
Vpx
mac251
, Vpx
mac239
, Vpx
Smm
, Vpx
drl1
, Vpx
ROD
, and Vpx
2A
with
huSAMHD1 (Figures 5A and 5B), suggesting a degradation of
huSAMHD1 in the presence of these Vpx alleles. To the contrary,
significant costaining between Vpx
Rcm-Ng
and Vpx
mnd2
with
huSAMHD1 was observed (Figure 5B), suggesting that no signif-
icant decrease of huSAMHD1 levels was induced by these
Vpx alleles. Intriguingly, Vpx/huSAMHD1 costaining was also
observed when Vpx
2B
was expressed, but this costaining was
cytoplasmic (Figures 5A and 5B), indicating a relocalization
of huSAMHD1 to the cytoplasm in the presence of Vpx
2B
.To
investigate whether this cytoplasmic localization of SAMHD1
might be an intermediate step en route toward degradation,
we treated differentiated THP-1 cells with pseudoparticles ex-
pressing Vpx
mac251
(VLP-Vpx) and followed SAMHD1 staining
over time by immunofluorescence (Figure 5C). Two hours after
VLP-Vpx treatment, a decrease of huSAMHD1 staining could
be observed, whereas 6 hr after VLP-Vpx treatment, the cells
displayed a heterogeneous SAMHD1 staining pattern, including
cells where no SAMHD1 signal could be detected and cells
where the SAMHD1 staining was cytoplasmic. Additionally,
Vpx was unable to degrade SAMHD1 when differentiated
THP-1 cells were treated with leptomycin B an inhibitor of protein
export (Figure S5). This strongly suggests that relocalization of
SAMHD1 from the nucleus to the cytoplasm might be an inter-
mediate step toward its final degradation.
We next analyzed the ability of these Vpx alleles to overcome
huSAMHD1 restriction activity. Differentiated THP-1 cells were
transduced so as to express Vpx alleles. Cells were infected
with HIV-LUC-G. Luciferase activity shows a significant increase
of infection by HIV-LUC-G after Vpx
mac251
, Vpx
mac239
, Vpx
Smm
,
Vpx
drl1
, Vpx
ROD
, and Vpx
2A
expression (Figure 5D). In this
context, Vpx
Rcm-Ng
, Vpx
mnd2
, and Vpx
2B
poorly affected
huSAMHD1 restriction. These experiments show a correlation
between Vpx ability to degrade huSAMHD1 and to overcome
its restriction activity.
To further investigate the importance of Vpx/SAMHD1 interac-
tion in this phenotype, we performed HA-immunoprecipitation
on whole-cell extracts from 293T cells coexpressing FLAG-
and HA-tagged Vpx and FLAG-huSAMHD1. We thereby show
that Vpx
mac251
, Vpx
Smm
, Vpx
drl1
, Vpx
ROD
, and Vpx
2B
interact
0
0,5
1
1,5
2
2,5
3
1
11
21
31
41
51
61
71
81
91
101
111
121
131
141
151
161
171
181
191
201
211
221
231
241
251
261
271
281
291
301
311
321
331
341
351
361
371
381
391
401
411
421
431
441
451
461
471
481
491
501
511
521
531
541
551
561
571
581
591
601
611
621
dN/dS
***
SAM
HD
*
BA
Theropithecus gelada*
Aotus trivirgatus*
Pygathrix nemaeus*
Cercopithecus diana*
Saimiri sciureus*
Macaca mulatta*
Ateles geoffroyi*
Papio hamadryas*
Cercocebus galeritus*
Homo sapiens*
Tarsius syrichta
Alouatta palliata*
Pongo pygmaeus*
Trachypithecus francoisi*
Mandrillus sphinx*
Tupaia belangeri
Allenopithecus nigroviridis*
Cebus apella*
Varecia variegata*
Otolemur garnettii
Gorilla gorilla
Callithrix jacchus*
Hylobates lar*
Nomascus leucogenys
Chlorocebus tantalus*
Saguinus oedipus*
Colobus angolensis*
Pongo abelii
Microcebus murinus*
Pan troglodytes*
Miopithecus talapoin*
Macaca fascicularis*
6
2
6
6
1
8
6
1
4
6
0
2
6
0
1
OWM
NWM
PRO
Hom.
Figure 3. Site-Specific Positive Selection Profile of SAMHD1 in 31 Primates
(A) The dN/dS ratio (u) of the 626 codons of the human SAMHD1 sequence was inferred under the M8 site model (Nielsen and Yang, 1998) implemented in
Codeml (Yang, 2007) from an alignment of 31 primates species with Tupaia belangeri as an outgroup. The dash line represents the dN/dS = 1 limit, above which
codons are inferred to be under positive selection. The 17 codons shown in red are those identified as being under positive selection with posterior probability
PP > 0.95 under M8 model. The four codons indicated by red stars are those identified using the more conservative M2a site model with PP > 0.95 (Zhang et al.,
2005). The position of the two functional SAM and HD domains is indicated.
(B) Amino acids in the C-terminal domain of SAMHD1 that are subject to strong positive selection and their distribution among primates is represented.
See also Figure S3.
Cell Host & Microbe
Evolution of the SAMHD1/Vpx Interaction
210 Cell Host & Microbe 11, 205–217, February 16, 2012 ª2012 Elsevier Inc.
with huSAMHD1, whereas only weak interaction was observed
with Vpx from SIV
Rcm-ng
and SIV
mnd2
(Figures 5E and 5F). Of
note, the interaction of SAMHD1 with Vpx
Smm
was weaker
than with Vpx
mac251
. It should be noted however that Vpx
2B
which interacts with huSAMHD1 will ultimately induce its degra-
dation and enhance HIV-1 infectivity but with slower kinetics
compared to Vpx
2A
. Indeed, when Vpx
2B
-expressing THP-1 cells
are harvested 96 hr after transduction, or when HeLa cells are
transiently transfected with huSAMHD1 and Vpx
2B
, protein
levels of huSAMHD1 are decreased at similar levels as when
cells express Vpx
ROD
or Vpx
2A
(data not shown).
Species-Specific Degradation of Primate SAMHD1
by Vpx
Given the differential activity of Vpx alleles to counteract
huSAMHD1 restriction activity, we finally tested the ability of
Vpx from different lentiviral strains to degrade SAMHD1 from
various primates by cotransfection of Vpx and SAMHD1 ex-
pressing plasmids in HeLa cells (Figure 6A). In agreement with
previous degradation assays performed in THP-1 cells (Laguette
et al., 2011), human SAMHD1 was significantly degraded in the
presence of Vpx
ROD
, Vpx
mac251
, and Vpx
Smm
but not in the
presence of Vpx
Rcm-Ng
and Vpx
mnd2
. A similar degradation
1
SAM
HD
53326180144
106
626
HD
AA
SAM
575
HD
SAM
595
HD
SAM
HD
SAM
611
SAM
HD
A
SAM
HD
A
SAM
HD
A
WT
ΔSAM
HD/AA
F575
F595
F611
614A
618A
626A
A
B
huSAMHD1:
Vpx
+++----
huSAMHD1
Vpx
Tubulin
+++---+-
DC
huSAMHD1:
Vpx
FLAG-IP
huSAMHD1
W.C.E.
Vpx
DDB1
Vpx:
++ + +
Vpx:
Vpx
Tubulin
Vpx
huSAMHD1
++ + + + +
huSAMHD1:
FLAG-IPW.C.E.
Figure 4. Interaction between Vpx
mac251
and huSAMHD1 through the C-Terminal Domain of SAMHD1
(A) Schematic representation of truncations and point mutants of huSAMHD1 that were engineered in the pOZ retroviral vector. All constructs were C-terminally
FLAG-tagged. The limits of SAM and HD domains are indicated.
(B) Amino acids 575 to 626 of SAMHD1 are required for Vpx
mac251
-induced hSAMHD1 degradation. FLAG-tagged WT-SAMH D1, SAMHD1-HD/AA, SAMHD1-
DSAM, and SAMHD1-F575 were transfected in 293T cells together or not with HA-tagged Vpx
mac251
. Whole-cell extracts were analyzed 48 hr later by immunoblot
using anti-FLAG, anti-HA, and anti-tubulin antibodies.
(C) Amino acids 595 to 626 of huSAMHD1 are involved in the interaction between Vpx
mac251
and huSAMHD1. Whole-cell extracts from 293T cells transfected to
express HA-Vpx
mac251
together with WT-SAMHD1, SAMHD1-F575, or SAMHD1-F595 were FLAG immunoprecipitated. After peptide elution, immunoprecipi-
tates and whole-cell extracts were analyzed by immunoblot with anti-FLAG, anti-HA, and anti-DDB1 antibodies.
(D) FLAG-tagged WT-SAMHD1, SAMHD1-F611, SAMHD1-S614A, SAMHD1-V618A, and SAMHD1-M626A were FLAG immunoprecipitated from 293T cells
expressing HA-Vpx
mac251
. Peptide-eluted immunoprecipitates were analyzed by western blot with anti-HA, anti-FLAG, and anti-tubulin antibodies.
See also Figure S4.
Cell Host & Microbe
Evolution of the SAMHD1/Vpx Interaction
Cell Host & Microbe 11, 205–217, February 16, 2012 ª2012 Elsevier Inc. 211
pattern was observed for SAMHD1 from another hominoid, the
gibbon, which is not naturally infected by SIV. Efficient degrada-
tion of rhesus macaque SAMHD1 was observed when coex-
pressed with Vpx from HIV-2
ROD
, and Vpx from two SIV strains
able to infect rhesus macaques (SIV
mac251
and SIV
Smm
). Addi-
tionally, protein levels of rhesus SAMHD1 were strongly
decreased in the presence of Vpx from SIV strains infecting
mangabeys (Vpx
Rcm-ng
) and mandrills (SIV
mnd2
). Consistently,
Vpx
mac251
and Vpx
Rcm-Ng
interacted with rhesus SAMHD1 in an
immunoprecipitation assay (Figure 6B). Similar to rhesus
SAMHD1, mangabey SAMHD1 was strongly degraded when
coexpressed with all Vpx proteins, including Vpx
Rcm-ng
and
Vpx
mnd2
. These results show that Vpx
Rcm-Ng
and Vpx
mnd2
can
induce the degradation of SAMHD1 proteins from Old World
monkeys (rhesus and mangabeys) and not from hominoids
(human and gibbon). Levels of SAMHD1 from the owl monkey,
a NWM that has not been exposed to SIV, were significantly
decreased in the presence of Vpx
ROD
, Vpx
mac251
, and Vpx
Smm
.
Interestingly, SAMHD1 from the gray mouse lemur, the most
distant primate as compared to humans, was resistant to
B
D
E
0
10
20
30
40
50
60
70
80
90
100
Cytoplasmic
Co-staining
No co-staining
% counted cells
C
mnd2 239smmRcm-ngdrl1 251
VpxSAMHD1 Merge Dapi
A
2A2B
V
px:
ROD
0 hr2 hr6 hr
VLP-Vpx
SAMHD1 SAMHD1+DAPI
+
-
Hs SAMHD1-F:
++++ +
F/H-Vpx:
+
HA-IPW.C.E.
Vpx
HuSAMHD1
Vpx
Tubulin
-
*
-----
HuSAMHD1
F
Vpx
HuSAMHD1
Vpx
Tubulin
HuSAMHD1
Hs SAMHD1-F:
F/H-Vpx:
HA-IPW.C.E.
0
10
20
30
40
50
60
70
80
Fold increase (luciferase activity)
Vpx:
Vpx:
Figure 5. Ability of Vpx Alleles from SIV and HIV-2 Strains to Degrade huSAMHD1
(A) THP-1 cells were transduced with retroviral vectors allowing expression of FLAG- and HA-tagged Vpx
mac251
, Vpx
mac239
, Vpx
Smm
, Vpx
Rcm-Ng
, Vpx
mnd2
, Vpx
drl1
,
Vpx
ROD
, Vpx
2A
, and Vpx
2B
. Forty-eight hours later, transduced cells were differentiated overnight on polylysine treated coverslips prior to immunostaining with
anti-SAMHD1 and anti-HA antibodies. Nuclei are stained in mounting media with DAPI. Arrows point at examples of cells displaying a representative staining.
(B) Vpx-positive cells from (A) were counted. Results are expressed as the percent of Vpx-positive cells where Vpx/SAMHD1 costaining, Vpx/SAMHD1 exclusion
and cytoplasmic SAMHD1 is observed.
(C) THP-1 cells differentiated on coverslips were treated with VLP-Vpx. Cells were fixed and stained with anti-SAMHD1 antibody at 0, 2, and 6 hr after VLP-Vpx
exposure. Nuclei are stained in mounting media with DAPI. Arrows point at examples of cells displaying a representative staining.
(D) Cells treated as in (A) were infected with HIV-LUC-G. Luciferase activity was measured 24 hr after infection and is expressed as fold increase luciferase activity
over untransduced parental THP-1 cells. Error bars represent the standard deviation from the mean.
(E) FLAG- and HA-tagged Vpx alleles from selected SIV strains were coexpressed with FLAG-tagged huSAMHD1 or empty vector in 293T cells.
Whole-cell extracts were subjected to HA immunoprecipitation prior to peptide elution and analy sis by immunoblot with anti-FLAG, anti-HA, and anti-tubulin
antibodies.
(F) FLAG- and HA-tagged Vpx alleles from HIV-2
ROD
and HIV-2B strains were coex pressed with FLAG-tagged huSAMHD1 or empty vector in 293T cells. Whole-
cell extracts were subjected to HA immunoprecipitation prior to peptide elution and analysis by immunoblot using anti-FLAG, anti-HA and anti-tubulin antibodies.
See also Figure S5.
Cell Host & Microbe
Evolution of the SAMHD1/Vpx Interaction
212 Cell Host & Microbe 11, 205–217, February 16, 2012 ª2012 Elsevier Inc.
degradation in the presence of all tested Vpx alleles. These data
highlight that Vpx alleles mediate the degradation of primate
SAMHD1 in a species-specific manner (Figure 6C).
DISCUSSION
Evolutionary analysis of host restriction factors (Strebel et al.,
2009) such as TRIM5a, APOBEC3G, and BST-2 highlight
episodes of selective pressure during primate evolution that
likely result from genetic conflict with retroviral elements (Ortiz
et al., 2009; Patel et al., 2011). Likewise, by analyzing the molec-
ular evolution of SAMHD1 in primates, and consistent with Lim
et al. (2012), we present evidence that SAMHD1 was subjected
to strong positive selection since catarrhines diverged from plat-
yrrhines. Unlike the above-cited host restriction factors, the
episodes of selection did not abrogate SAMHD1 restriction
activity toward HIV-1, since SAMHD1 orthologs from very distant
primates exhibit potent anti-HIV-1 activity similar to that of
huSAMHD1. However, certain lentiviruses have evolved means
of counteracting SAMHD1 restriction through the viral auxiliary
protein Vpx. Here, we harnessed positive selection analysis
as a tool to identify the C-terminal domain of huSAMHD1 as
required for interaction with Vpx
mac251
and subsequent targeting
of SAMHD1 to proteasomal degradation. We further mapped the
amino acid M626 of SAMHD1 as required for the Vpx/SAMHD1
interaction. Finally, we show that, in contrast to SAMHD1 broad
range activity, the ability of Vpx to induce the degradation of
primate SAMHD1 is species specific.
Inference of Catarhinni, Platyrhinni, Simiiformes, and primate
ancestral SAMHD1 (Figure S3) illustrates the accumulation of
SAMHD1
Tubulin
Vpx
SAMHD1
Tubulin
Vpx
A
B
RhSAMHD1-F:
++
RhSAMHD1
--
Vpx
Vpx
Tubulin
RhSAMHD1
C.E.
HA-IP
F/H-Vpxmac251:
F/H-VpxRcm-Ng:
--++
-+-+
Hu Rh
Mng
Owl
Gml
Gib
Hominoids OWM NWM/prosimian
SAMHD1
Vpx
+
-
+++++ +
-
+++++ +
-
+++++
HIV-2
Vpx (ROD)
SIV
mac251
Vpx
SIV
smm
Vpx
SIV
rcm-NG
Vpx
SIV
mnd2
Vpx
Human
Gibbon
Rhesus
Mangabey
Hominoids
OWM
Owl
monkey
NWM
Prosimian
Gray
mouse
lemur
C
Figure 6. Vpx-Induced SAMHD1 Degradation Is Species Specific
(A) SAMHD1 expression patterns in the presence of Vpx from HIV-2 and SIV strains. FLAG- and HA-tagged SAMHD1 proteins from a selected panel of primates
were coexpressed with various FLAG- and HA-tagged Vpx proteins in HeLa cells. Whole-cell extracts were analyzed 30 hr after transfection by immunoblot with
anti-HA and anti-tubulin antibodies. Hu, human; Gib, gibbon; Rh, rhesus macaque; Mng, mangabey; OWM, Old World monkeys; Owl, owl monkey; NWM, New
World monkeys; Gml, gray mouse lemur.
(B) Vpx
Rcm-Ng
and Vpx
mac251
interact with rhesus SAMHD1. FLAG- and HA-tagged Vpx alleles were coexpressed with FLAG-tagged rhesus SAMHD1 or empty
vector in 293T cells. Whole-cell extracts were subjected to HA immunoprecipitation prior to peptide elution and analysis by immunoblot with anti-FLAG, anti-HA,
and anti-tubulin antibodies.
(C) Schematic representation summarizing the data obtained showing that Vpx targeting of SAMHD1 is species specific. HIV-2 Clade B Vpx (discontinuous arrow)
degrades huSAMHD1 but with slower kinetics compared to HIV-2
ROD
Vpx.
Cell Host & Microbe
Evolution of the SAMHD1/Vpx Interaction
Cell Host & Microbe 11, 205–217, February 16, 2012 ª2012 Elsevier Inc. 213
amino acid changes along SAMHD1 sequence over 60 million
years of evolution. Adaptation of the SAMHD1 sequence prior
to diversification of the Catarrhini coincides with multiple events
of invasion of the primate lineage by endogenous retroviruses
such as HERV-K, HERV-H, HERV-E, HERV-W, and ERV-9
(Bannert and Kurth, 2006 ). In this perspective, it will be important
to investigate whether endogenous retroviruses played a role in
SAMHD1 adaptation and whether SAMHD1 may affect other
retroviruses. In agreement, Vpx may alleviate a monocyte-
specific restriction of murine leukemia virus infection (Jarros-
son-Wuilleme et al., 2006; Kaushik et al., 2009). This notwith-
standing, the accumulation of positively selected sites clustered
in the N-terminal and C-terminal of the protein possibly results
from lineage-specific adaptation of SAMHD1, after the split
between hominoids and OWMs that occurred around 18 million
years ago and may therefore result from genetic conflict between
SAMHD1 and more recent viruses such as lentiviruses. Indeed,
the most recent dating estimates the age of lentiviruses to
a minimum of 12 million years (Katzourakis et al., 2007; Kecke-
sova et al., 2009).
One of the hallmarks of positively selected restriction factors in
primates is their species-specific antiretroviral activity (Mariani
et al., 2003; McNatt et al., 2009; Song et al., 2005). However,
we observed that primate SAMHD1 orthologs distant by
60 million years (divergence human/gray mouse lemur) were
able to block HIV-1. Thus, despite selective pressure occurring
over millions of years, SAMHD1 restrictive potential against
HIV-1 appears to be evolutionarily conserved.
Genetic conflict between host restriction factors and viruses is
predicted to lead to rapid selection of mutations that alter amino
acid composition of both actors, especially at positions that
affect protein-protein interaction. This type of genetic conflict
has been shown to occur between APOBEC3G and Vif (Sawyer
et al., 2004 ), TRIM5a and the viral capsid (Stremlau et al., 2005 ),
BST-2 and Vpu, Nef, or Env (McNatt et al., 2009). In the case of
APOBEC3G, TRIM5a, and BST-2, domains involved in direct
interaction with retroviral factors are enriched in positively
selected sites (Ortiz et al., 2009). Similarly, analysis of positive
selection on SAMHD1 sequence reveals a cluster of sites in
the C-terminal domain under selective pressure. Deletion of
the C terminus of huSAMHD1 results in loss of interaction with
Vpx
mac251
and subsequent loss of SAMHD1 degradation. This
suggests that Vpx may have dictated the evolution of the
C-terminal domain of SAMHD1. Mutational analysis of the sites
under selection in C terminus reveals that at least M626 of
huSAMHD1 is important for the interaction with Vpx
mac251
.
M626 is one of the few amino acids of SAMHD1 that vary in
a lineage-specific manner in primates. Position 626 may be
important for the interaction of Vpx with cognate SAMHD1,
considering the species-specificity of Vpx-induced SAMHD1
degradation. However, both owl monkey and gray mouse lemur
SAMHD1 harbor a V626, but only owl monkey SAMHD1 is effi-
ciently degraded by Vpx
mac251
. This raises the possibility that
position 626 is not the only site recognized by Vpx. Indeed,
Lim et al. (2012) show that positively selected residues within
the N-terminal domain of SAMHD1 determine both binding and
susceptibility to Vpx. Additional traces of positive selection are
seen in the N-terminal domain of SAMHD1, comprising the
SAM domain predicted to mediate SAM/SAM, protein/protein,
or protein/RNA interaction. It is therefore possible that SAMHD1
does not solely interact with Vpx and suggests that SAMHD1
may target another viral component to exert its restriction
activity. Interestingly, position 256, within the HD domain, was
also detected to be under positive selection. It will be of impor-
tance to determine whether this residue is involved in SAMHD1
function.
Additionally, we also show that Vpx
2A
and Vpx
2B
differ in their
ability to degrade SAMHD1. Indeed, Vpx
2B
causes SAMHD1
degradation at a slower rate than Vpx
2A
. This feature allows for
a mechanistic insight into Vpx-induced SAMHD1 degradation:
Vpx seems to cause relocalization of SAMHD1 to the cytoplasm
prior to routing toward the proteasome machinery. Since Vpx
2B
efficiently interacts with SAMHD1, one may speculate that the
interaction of Vpx
2B
with the E3-ligase complex involved in
SAMHD1 degradation and its export from the nucleus are less
efficient than those of Vpx
2A
. The different clades of HIV-2 may
have originated from independent cross-species transmission
event (Lemey et al., 2003). Whether the differential kinetics of
Vpx-mediated enhancement of DC infection after infection with
HIV-2 from clades A or B affects pathogeny, remains to be
explored.
Since the viral auxiliary protein Vpx is not shared by all lentivi-
ruses, this raises concerns of the necessity to bypass this restric-
tion for efficient viral spread. In particular, pandemic HIV-1
strains have not evolved means to induce SAMHD1 degradation,
whereas nonpandemic HIV-2 induces SAMHD1 degradation
through its Vpx. A consequence of HIV-1 not possessing Vpx
is the inability of this virus to infect DCs, particularly at an early
stage of viral transmission when viral loads are very low. Whether
DCs from HIV-2 patients are productively infected in the context
of a natural infection is unknown. Interestingly, phylogenetic
studies have traced the origin of pandemic and nonpandemic
HIV-1 (group M and groups N, O, and P, respectively) infections
to cross-species transmissions from chimpanzees and gorillas
(Keele et al., 2006). SIV
cpz
has been shown to originate from
a probable recombination of lentiviruses from red-capped
mangabeys (SIV
Rcm
) and greater spot-nosed monkeys (SIV
gsn
)
(Bailes et al., 2003). In this process, it appears that the vpx
gene of SIV
Rcm
was lost. Infection by SIV
cpz
in chimpanzees
causes symptoms closely related to AIDS (Keele et al., 2009).
Whether this ORF was lost before the transmission to chimpan-
zees is unknown. This observation is to be examined in the light
of previous reports hypothesizing that vpx would have originated
from gene duplication of an ancestral vpr gene or by acquisition
of a heterolougous vpr allele (Tristem et al., 1992; Tristem et al.,
1990). HIV-2 infection originated from an independent cross-
species transmission of SIV
Smm
from Sooty mangabeys (Hirsch
et al., 1989; Wertheim and Worobey, 2009) that possess a vpx
gene. One may question whether the presence of Vpx may
have represented an advantage favoring the cross-species
transmission. However, HIV-2 infection is characterized by its
nonpandemicity and the decline in its prevalence as compared
to the increasing prevalence of HIV-1 (Tebit and Arts, 2011).
Nonetheless, Vpx seems to be dispensable for persistence and
spread in humans. The restriction factor SAMHD1 appears to
bridge anti-viral responses and innate immune responses. The
interplay between SAMHD1 and Vpx reflects how the immune
system adapts to the reciprocal constraints exerted by viruses
Cell Host & Microbe
Evolution of the SAMHD1/Vpx Interaction
214 Cell Host & Microbe 11, 205–217, February 16, 2012 ª2012 Elsevier Inc.
and the host, especially in the light of restriction factors being
involved in the triggering of innate immune responses.
EXPERIMENTAL PROCEDURES
Evolutionary Analyses and Ancestral Sequence Reconstruction
The CDS amino acid alignment containing the 34 sequences available for
SAMHD1 was retrieved from the OrthoMaM database (Ranwez et al., 2007).
Other 786 1:1 orthologous CDS amino acid alignments for which the same
34 taxa were available were also downloaded from OrthoMaM. Ambiguously
aligned sites were excluded using Gblocks default parameters (Castresana,
2000). Maximum likelihood branch lengths were then optimized for each alig n-
ment by using maximum likelihood under the LG+G8+F model on a fixed
topology with PhyML 3 (Guindon et al., 2010). The same branch length optimi-
zation protocol was applied to the concatenation of the 786 genes but using
RAxML v7.2 .8 (Stamatakis, 2006).
The 32-taxa data set was constructed from the 25 newly obtained SAMHD1
sequences and seven sequences retrieved from EnsEMBL (Table S1). The
alignment length was restricted to the length of the human SAMHD1 sequence
(626 codons) by excluding all sites containing gaps in nonhuma n sequences.
Maximum likelihood phylogenetic reconstruction was performed from this
nucleotide alignment under the GTR+GAMMA model with RAxML.
Most of the analyses aiming at describing the selective constraints acting on
SAMHD1 in primates were conducted using the site-specific, branch-specific,
and branch-site models for detecting positive selection implemented in the
CODEML program of the PAML 4.4 package (Yang, 2007). All calculations
were performed using the ML topology previously inferred by RAxML.
The Bayesian reconstruction of dN/dS variation along the primate SAMHD1
ML phylogeny was conducted using the dsom procedure implemented in the
CoEvol program (Lartillot and Poujol, 2011). We used the seven calibrations
proposed by Perelman et al. (2011) that were compatible with our taxon
sampling to infer divergence times. The values of the three life-history traits
incorporated in the analysis (body mass, longevity, and maturity) were
obtained from the PanTheria database. Two independent MCMC were run
for a total of 100,000 cycles sampling points every ten cycles. The first 1,000
points were then excluded as the burnin and inferences were made from the
remaining 9,000 sampled points.
Expression Constructs and Plasmids
All SAMHD1 coding sequence s were C-terminally tagged with FLAG and HA
by cloning into the previously described MMLV-based retroviral vector
(Kumar et al., 2009). For immunoprecipitation experiments, selected SAMHD1
alleles were subcloned into a similar vector except that the HA tag was
removed. Constructs expressing FLAG- and HA-tagged Vpx
mac251
, Vpx
ROD
,
Vpx
RCM-NG
, and Vpx
RCM-GAB
were previously described (Laguette et al.,
2011). Vpx alleles from SIVmac239, SIVsm, SIVmnd2, SIVdrl1, HIV-2A, and
HIV-2B were synthesized by GenScript (Table S2) and subcloned in pOZ
vector (Nakatani and Ogryzko, 2003). For immunoprecipitation experiments,
Vpx
mac251
was further subcloned in pOZvector where the FLAG tag was
deleted.
N-terminal (SAMHD1DSAM) and C-terminal (SAMHD1-F575, SAMHD1-
F595, SAMHD1-F611) truncations in SAMHD1 were generated with the
phusion enzyme (Finnzym). Point mutations in the SAMHD1 (SAMHD1-
S614A, V618A, and M626A ) sequence were introduced using the Quickchange
lightning kit (Agilent Technologies) according to the manufacturer’s recom-
mendations. SAMHD1-HD/AA and SAMHD1-R mutants, HIV-LUC, VSV-G,
MMLV packaging, A-MLV envelope, and shRNA constructs were previously
described (Laguette et al., 2011 ).
pBRSIVmac239_nef+_IRES_eGFPd5 was generated by introducing a MluI
restriction site just upstream of the unique SmaI site located downstream
of the nef ORF in pBRSIVmac239nef+TPId5 (Mu
¨
nch et al., 2001). Next,
a SmaI-IRES-eGFP-MluI fragment was inserted. SIVmac vpx STOP was
generated by mutating the start codon of vpx to ACG and introducing a stop
codon (TGA) at position 2 without changing the reading frame of vif. These
mutations were introduced by PCR site-directed mutagenesis using the
Phire polymerase (Finnzymes) and cloned into the SIVmac proviral contruct
via PacI/SphI.
Immunopurification
For immunoprecipitation experiments, whole-cell extracts were prepared with
10% glycerol, 0.5% triton, 150 mM NaCl, 10 mM KCL, 1 .5 mM MgCl2, 0.5 mM
EDTA, 10 mM b -mercaptoethanol, and 0.5 mM PMSF. When FLAG-tagged
SAMHD1 was immunoprecipitated, extracts were incubated with anti-FLAG
antibody conjugated agarose beads (Sigma), and the bound polypeptides
were eluted with Flag peptide (Sigma). When HA-tagged Vpx was immunopre-
cipitated, extracts were incubated with anti-HA antibody conjugated agarose
beads (Santa Cruz), and the bound polypeptides were eluted with Flag peptide
(Roche) under native conditions.
Immunofluoresence
THP-1 cells were transduced with Vpx expressing retroviral constructs 48 hr
prior to 16 hr differentiation on polylysine treated coverslips. Alternatively,
THP-1 cells were differentiated on polylysine treated coverslips prior to VLP-
Vpx treatment. HeLa cells were differentiated on coverslips prior to transfec-
tion with SAMHD1 constructs. Fixation was performed using PBS with 3%
paraformaldehyde and 2% sucrose. Permeabilization was achieved with
0.5% Triton X-100, 20 mM Tris (pH 7.6), 50 mM Nacl, 3 mM MgCl2, and
300 mM sucrose. Wash steps and antibody incubation steps were performed
in PBS-0.1%Tween. For SAMHD1 visualization, anti-SAMHD1 (Abcam) was
used (1:1000 dilution). For Vpx visualization, anti-HA from Covance was
used (1:1000 dilution). Secondary antibodies were purchased from Invitrogen.
Nuclei were stained with DAPI in mounting media (Vectashield; Vector Labs)
and images were collected on a Leica DM6000 microscope.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
five figures, and two tables and can be found with this article online at
doi:10.1016/j.chom.2012.01.007.
ACKNOWLEDGMENTS
We are grateful to Christine Goffinet for critical reading of the manuscript,
Sabine Chabalier-Laurent, Xuehua Li, Susanne Engelhart, Raquel Martinez,
and Simon Meister for excellent assistance. We are also thankful to the
many zoos and primate centers for providing the simian specimens, including
the Duke Lemur Center (DLC). N.L. is recipient of SIDACTION fellowship. Work
in M.B.’s laboratory was supported by ERC (250333), ANRS, SIDACTION, and
FRM ‘Equipe labe
´
llise
´
e FRM’ to M.B. J.S. was supported by the Research
Foundation Flanders (FWO, 1.2.627.07.N.01.). N.R. and A.T. are supported
by the Swiss National Science Foundation (grant 31003A_132863/1). Work
in F.K.’s laboratory was supported by the Deutsche Forschungsgemeinschaft.
Use of trade names is for identification only and does not imply endorsement
by the U.S. Department of Health and Human Services, the Public Health
Service, or the Centers for Disease Control and Prevention. The findings and
conclusions in this report are those of the authors and do not necessarily
represent the views of the Centers for Disease Control and Prevention. This
is contribution ISEM 2012-015 of the Institut des Sciences de l’Evolution de
Montpellier. We are grateful to Montpellier RIO Imaging for help with the
microscopy analyses. This is DLC publication #1214.
Received: November 4, 2011
Revised: December 8, 2011
Accepted: January 12, 2012
Published online: February 2, 2012
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    • "Of note, preincubation of murine BMDCs with SIV virus-like particles (VLP) containing the HIV-2/ SIV accessory protein Vpx did not counteract the antiviral activity of muSAMHD1 by inducing the degradation of endogenous SAMHD1 (data not shown). This is in line with previous sequence analysis and infection experiments showing that exogenously overexpressed muSAMHD1 is not degraded by Vpx [18, 19, 23], Next, we differentiated human U937 myeloid cells containing the mouse SAMHD1 isoforms 1 or 2 or an empty control vector into macrophage-like cells by incubating the cells with the phorbol ester PMA. The murine SAMHD1 splice variants differ in their C-terminal tail. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Human SAMHD1 is a triphosphohydrolase that restricts the replication of retroviruses, retroelements and DNA viruses in noncycling cells. While modes of action have been extensively described for human SAMHD1, only little is known about the regulation of SAMHD1 in the mouse. Here, we characterize the antiviral activity of murine SAMHD1 with the help of knockout mice to shed light on the regulation and the mechanism of the SAMHD1 restriction and to validate the SAMHD1 knockout mouse model for the use in future infectivity studies. Results: We found that endogenous mouse SAMHD1 restricts not only HIV-1 but also MLV reporter virus infection at the level of reverse transcription in primary myeloid cells. Similar to the human protein, the antiviral activity of murine SAMHD1 is regulated through phosphorylation at threonine 603 and is limited to nondividing cells. Comparing the susceptibility to infection with intracellular dNTP levels and SAMHD1 phosphorylation in different cell types shows that both functions are important determinants of the antiviral activity of murine SAMHD1. In contrast, we found the proposed RNase activity of SAMHD1 to be less important and could not detect any effect of mouse or human SAMHD1 on the level of incoming viral RNA. Conclusion: Our findings show that SAMHD1 in the mouse blocks retroviral infection at the level of reverse transcription and is regulated through cell cycle-dependent phosphorylation. We show that the antiviral restriction mediated by murine SAMHD1 is mechanistically similar to what is known for the human protein, making the SAMHD1 knockout mouse model a valuable tool to characterize the influence of SAMHD1 on the replication of different viruses in vivo.
    Full-text · Article · Dec 2015
    • "As a proof-of-principle, TRIM5α and APOBEC3G were identified in the screen. Consistent with previous reports [17,18], BST2 and SAMHD1 reached dN/dS ratios of 0.78 and 0.49, respectively; therefore, they were not shortlisted on the basis of global dN/dS ratios, although they displayed codon-specific positive selection. To also consider factors displaying only codon-specific positive selection, we additionally performed a second screen and ranking approach that are described below. "
    [Show abstract] [Hide abstract] ABSTRACT: Known antiretroviral restriction factors are encoded by genes that are under positive selection pressure, induced during HIV-1 infection, up-regulated by interferons, and/or interact with viral proteins. To identify potential novel restriction factors, we performed genome-wide scans for human genes sharing molecular and evolutionary signatures of known restriction factors and tested the anti-HIV-1 activity of the most promising candidates. Our analyses identified 30 human genes that share characteristics of known restriction factors. Functional analyses of 27 of these candidates showed that over-expression of a strikingly high proportion of them significantly inhibited HIV-1 without causing cytotoxic effects. Five factors (APOL1, APOL6, CD164, TNFRSF10A, TNFRSF10D) suppressed infectious HIV-1 production in transfected 293T cells by >90% and six additional candidates (FCGR3A, CD3E, OAS1, GBP5, SPN, IFI16) achieved this when the virus was lacking intact accessory vpr, vpu and nef genes. Unexpectedly, over-expression of two factors (IL1A, SP110) significantly increased infectious HIV-1 production. Mechanistic studies suggest that the newly identified potential restriction factors act at different steps of the viral replication cycle, including proviral transcription and production of viral proteins. Finally, we confirmed that mRNA expression of most of these candidate restriction factors in primary CD4+ T cells is significantly increased by type I interferons. A limited number of human genes share multiple characteristics of genes encoding for known restriction factors. Most of them display anti-retroviral activity in transient transfection assays and are expressed in primary CD4+ T cells.
    Full-text · Article · May 2015
    • "Restriction factors and accessory proteins are engaged in an evolutionary ''molecular arms race'' consisting of multiple rounds of host adaptation, virus counteraction, and host readaptation , resulting in accumulation of amino acid changes in restriction factor-accessory protein interaction interfaces (Daugherty and Malik, 2012). In the lentiviral Vpx/Vpr accessory proteins, analyses of positively selected residues and subsequent functional studies have demonstrated the occurrence of several significant specificity changes during adaptation to primate hosts (Laguette et al., 2012; Lim et al., 2012). It is proposed that originally a subset of Vpr proteins acquired the capability to induce cullin-4/DCAF1-dependent proteasomal degradation of SAMHD1. "
    [Show abstract] [Hide abstract] ABSTRACT: The SAMHD1 triphosphohydrolase inhibits HIV-1 infection of myeloid and resting T cells by depleting dNTPs. To overcome SAMHD1, HIV-2 and some SIVs encode either of two lineages of the accessory protein Vpx that bind the SAMHD1 N or C terminus and redirect the host cullin-4 ubiquitin ligase to target SAMHD1 for proteasomal degradation. We present the ternary complex of Vpx from SIV that infects mandrills (SIVmnd-2) with the cullin-4 substrate receptor, DCAF1, and N-terminal and SAM domains from mandrill SAMHD1. The structure reveals details of Vpx lineage-specific targeting of SAMHD1 N-terminal "degron" sequences. Comparison with Vpx from SIV that infects sooty mangabeys (SIVsmm) complexed with SAMHD1-DCAF1 identifies molecular determinants directing Vpx lineages to N- or C-terminal SAMHD1 sequences. Inspection of the Vpx-DCAF1 interface also reveals conservation of Vpx with the evolutionally related HIV-1/SIV accessory protein Vpr. These data suggest a unified model for how Vpx and Vpr exploit DCAF1 to promote viral replication. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · Apr 2015
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