HIV-1 Vpr activates the G2checkpoint through manipulation of the ubiquitin proteasome system

Zimmerman, Dehart, Ardon Orly, Monteiro-Filho, Argañaraz, Planelles, Planelles Vicente

Description

HIV-1 Vpr is a viral accessory protein that activates ATR through the induction of DNA replication stress. ATR activation results in cell cycle arrest in G 2 and induction of apoptosis. In the present study, we investigate the role of the ubiquitin/proteasome system (UPS) in the above activity of Vpr. We report that the general function of the UPS is required for Vpr to induce G 2 checkpoint activation, as incubation of Vpr-expressing cells with proteasome inhibitors abolishes this effect. We further investigated in detail the specific E3 ubiquitin ligase subunits that Vpr manipulates. We found that Vpr binds to the DCAF1 subunit of a cullin 4a/DDB1 E3 ubiquitin ligase. The carboxy-terminal domain Vpr(R80A) mutant, which is able to bind DCAF1, is inactive in checkpoint activation and has dominant-negative character. In contrast, the mutation Q65R, in the leucine-rich domain of Vpr that mediates DCAF1 binding, results in an inactive Vpr devoid of dominant negative behavior. Thus, the interaction of Vpr with DCAF1 is required, but not sufficient, for Vpr to cause G 2 arrest. We propose that Vpr recruits, through its carboxy terminal domain, an unknown cellular factor that is required for G 2 -to-M transition. Recruitment of this factor leads to its ubiquitination and degradation, resulting in failure to enter mitosis.

Informations

• Publié par: biomed
• Publié le: 01 janvier 2007
• Nombre de lectures: 12
• Langue: English

Extrait

BioMed CentralVirology Journal
Open AccessResearch
HIV-1 Vpr activates the G checkpoint through manipulation of the 2
ubiquitin proteasome system
†1 †1 1Jason L DeHart , Erik S Zimmerman , Orly Ardon , Carlos MR
Monteiro2 2 1Filho , Enrique R Argañaraz and Vicente Planelles*
1Address: Division of Cell Biology and Immunology, Department of Pathology, University of Utah School of Medicine, 15 North Medical Drive
2East #2100 – Room 2520, Salt Lake City, UT 84112, USA and Laboratório de Farmacologia Molecular (CP 04536), Faculdade de Saude,
Universidade de Brasília, 70919-970 Brasília, DF, Brazil
Email: Jason L DeHart - [e-mail]; Erik S Zimmerman - [e-mail];
Orly Ardon - [e-mail]; Carlos MR Monteiro-Filho - [e-mail];
Enrique R Argañaraz - [e-mail]; Vicente Planelles* - [e-mail]
* Corresponding author †Equal contributors
Published: 8 June 2007 Received: 4 June 2007
Accepted: 8 June 2007
Virology Journal 2007, 4:57 doi:10.1186/1743-422X-4-57
This article is available from: http://www.virologyj.com/content/4/1/57
© 2007 DeHart et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
HIV-1 Vpr is a viral accessory protein that activates ATR through the induction of DNA replication
stress. ATR activation results in cell cycle arrest in G and induction of apoptosis. In the present2
study, we investigate the role of the ubiquitin/proteasome system (UPS) in the above activity of Vpr.
We report that the general function of the UPS is required for Vpr to induce G checkpoint2
activation, as incubation of Vpr-expressing cells with proteasome inhibitors abolishes this effect.
We further investigated in detail the specific E3 ubiquitin ligase subunits that Vpr manipulates. We
found that Vpr binds to the DCAF1 subunit of a cullin 4a/DDB1 E3 ubiquitin ligase. The
carboxyterminal domain Vpr(R80A) mutant, which is able to bind DCAF1, is inactive in checkpoint
activation and has dominant-negative character. In contrast, the mutation Q65R, in the leucine-rich
domain of Vpr that mediates DCAF1 binding, results in an inactive Vpr devoid of dominant negative
behavior. Thus, the interaction of Vpr with DCAF1 is required, but not sufficient, for Vpr to cause
G arrest. We propose that Vpr recruits, through its carboxy terminal domain, an unknown cellular2
factor that is required for G -to-M transition. Recruitment of this factor leads to its ubiquitination2
and degradation, resulting in failure to enter mitosis.
Cell cycle progression is tightly regulated by several mech-Background
The HIV-1 encoded viral protein R induces cell cycle arrest anisms, including orchestrated destruction of cell cycle
and apoptosis through activation of the serine/threonine mediators, their phosphorylation/de-phosphorylation
kinase known as the ataxia telangiectasia-mutated and and their subcellular localization. Destruction of cell cycle
Rad3-related (ATR) protein [1,2]. Vpr activates ATR by regulators is typically mediated by the proteasome and
inducing replication stress, a cellular condition that involves polyubiquitination by E3 ubiquitin ligases. The
occurs in dividing cells as a consequence of deoxyribonu- existence of a connection between proteasomal
degradacleotide depletion, stalled replication forks, or ultraviolet tion of cell cycle regulators and ATR activation is
exemplilight-induced DNA damage. How Vpr induces replication fied in several instances involving Cdt1 [3-5] and Chk1
stress remains uncertain. [6] among others.
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Certain viral proteins are known to bind to the substrate involved, we next examined the potential E3 ligase
comspecificity subunits of E3 ligases to redirect specificity to ponents that are relevant to Vpr.
non-cognate targets. Examples of these viral proteins
include hepatitis B protein X [7], human papilloma virus Affinity chromatography and mass spectrometry identify
E6 [8], simian virus 5 V protein [9,10], HIV-1 Vif [11-13], DCAF1 as a potential interactor of Vpr
and HIV-1 Vpu [14]. In the present study, we examined in In an effort to identify cellular proteins that may interact
detail the potential role of the UPS in the ability of HIV-1 with Vpr to mediate its function, we performed affinity
Vpr to induce G arrest. chromatography followed by mass spectrometry. 293FT2
cells were transfected with a vector encoding a
hexa-histiResults and Discussion dine and hemagglutinin-tagged Vpr construct
(pHR-HisProteasome inhibitors relieve Vpr-induced G arrest HA-VPR-IRES-GFP), or mock-transfected, and then lysed2
Several lines of evidence suggest a possible functional at 24 hours. Lysates were bound to a Ni-NTA agarose
colinteraction of Vpr with the UPS. First, a protein known as umn. Bound proteins were eluted and then
immunopreRIP, that was discovered as an interaction partner of Vpr cipitated with an anti-HA antibody followed by protein G
[15], was recently shown to be part of a family of WD- agarose beads, boiled and resolved on SDS-PAGE. The
repeat proteins that are found in association with cullin resulting gel was silver-stained (Figure 2, panel A). We
4a/DDB1 E3 ubiquitin ligases [9]. Accordingly, RIP was observed three high-molecular weight bands (labeled "a,
recently renamed DDB1-Cul4A-associated factor-1, b and c") present in the Vpr lane but not in the control
DCAF1 [9]. Second, Vpr was recently found to induce deg- lane (Figure 2, panel A). Bands a, b, and c were excised,
radation of uracil-N-glycoslylase (UNG) through the UPS trypsin digested, and analyzed by mass spectrometry.
[16]. Finally, post transcriptional silencing of the dam- Band c was identified as DCAF1 [9], and was recently
aged DNA-binding protein 1 (DDB1) leads to cell cycle reported by Le Rouzic et al. to interact with Vpr [20].
arrest at the G -to-M transition [3]. Therefore, we set out Bands a and b could not be identified.2
to directly evaluate the role of the UPS in Vpr induced G2
arrest. We resorted to two different methods of
proteasome inhibition: incubation with epoxomicin, and
overexpression of a dominant-negative ubiquitin mutant,
Ub(K48R) [17] that blocks formation of polyubiquitin
chain conjugates. Cells were either incubated with
epoxomicin, DMSO, or transfected with Ub(K48R) or empty
vector. To induce Vpr expression, we transduced HeLa
cells with the Vpr-expressing lentivirus vector,
pHR-VPRIRES-GFP [2,18], and analyzed the cell cycle profile 48
post transduction. The vector pHR-VPR-IRES-GFP
expresses Vpr in the absence of all other HIV-1 genes, and
also expresses GFP via an internal ribosome entry site
[19]. For simplicity, we will refer to this lentiviral vector as
pHR-VPR. Throughout this work, we measured GFP
expression by flow cytometry and HA-Vpr expression by
WB, to verify that levels of infection with lentiviral vectors
were not affected by the various treatments (inhibitors,
siRNAs and dominant-negative constructs).
Incubation with epoxomicin induced a small, basal level
of G arrest in non-Vpr expressing cells. Strikingly, how-2
ever, epoxomicin incubation dramatically relieved
Vprinduced G arrest (Figure 1; cell cycle profile data are pre-2
sented in Additional file 1). In agreement with the
epoxomicin results, over-expression of Ub(K48R) also very
Role of the ubiquitin prFigure 1arrest oteasome system in Vpr-induced G2
effectively abolished the induction of G arrest in Vpr-2 2
expressing cells (Figure 1). Therefore, we conclude that arrest. Incubation with epoxomicin or overexpression of
Vpr function requires the activity of the UPS. On the other Ub(K48R) block Vpr induced G arrest when induced by Vpr, 2
hand, because the above proteasome inhibitors do not but not when induced by the topoisomerase inhibitor,
etoposide.provide any information on the specific ubiquitin ligases
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aphidicolin both activate ATR, our results suggest,
although do not demonstrate, the possibility that the
presence of DCAF1 may be normally required for ATR
activation.
DCAF1 brings HIV-1 Vpr in association with Cullin4/DDB1
Since the presence of DCAF1 is required for Vpr function,
we decided to test whether Vpr interacts with DCAF1. In
addition, because DCAF1 is known to function in the
context of DDB1 [9,23,24], which bridges DCAF1 to cullin 4,
we also asked whether Vpr can be found in association
with DDB1.
We transfected a Flag-DCAF1 construct along with either
HA-Vpr or HA-Vpr(R80A), a Vpr mutant that is incapable
Figure 2Role of DCAF1 in Vpr-induced G arrest2 of inducing G arrest [2,18,25] and, 48 hours later, we2 Role of DCAF1 in Vpr-induced G arrest. A. Identification of 2
immunoprecipitated Flag-DCAF1 from cell extracts.Vpr-interacting proteins by affinity chromatography followed
When immunoprecipitates obtained with anti-HA anti-by mass spectrometry; band labeled as "