A Lost Opportunity
For all those that attacked Miller, Andy and myself. This was the research we were looking into and how Xpr1 might be involved in neuromuscular pathology seen in CFS and may have relevance to neurologic disease induced by MCF retroviruses in mice. We had no intention for the sole purpose of finding xmrv in patients to determine who was positive or negative, conducting replication studies nor undermining the WPI or Mikovits as mentioned ad nauseum over and over again with little affect. We wanted to get past that and investigate some interesting behavior with the Xpr1. This was the main purpose and second stage of our research project which could not be disclosed. We were using sophisticated assays which are confidential to find more than just xmrv. This assay's are IP and cannot be disclosed.
Miller found something that could explain some of the symptoms that relate to ME in patients. We wanted to explore the involvement of the Xpr1 receptor in ME/CFS patients. It was mentioned time and time again our research was going past the point on proving the existence of xmrv but we were trying to determine the cause and find a treatment protocol that would negate the effects found in our research. Unfortunately, our research project was sabotage by those who now no longer subscribe to xmrv. It is kind of sad.
It is unfortunate that Lulu made so many libelous comments on postings in the UK forum and recently Jamie's blog about our intent. It was my decision for one purpose only. After reading the horror stories concerning the treatment of UK patients, I thought it would be a beacon of hope to those patients. Big mistake. In addition, Cort conducted a 2 hour question and answer question with Miller that he was going to post on the front page section of his forum which was scrub. I was going to post the information on Cort's forum as we were looking for patients close by and not in the UK. The posting in the UK forum was FYI only. Because of the uproar, the research was stopped.
I suppose I will come under attack for this posting as well. As Peterson alluded to, many researchers are turning away from helping this patient community. To project the medical policy that is occurring in another country as being the same as in this country with all its intended conspiracies is absurd. We are under a different administration with different administrator, director and department heads. Retrovirologists want to find another retro virus as it means greater funding for their department. Otherwise budget cuts and layoffs are the norm. Most institutions receive their support from private donations within their own community and are not Federal agencies. Of all the meetings, talks and face to face, one on ones with leading retrovirologist not one indicated or demonstrated any sort of conspiratorial behavior, just the opposite was true.
1
1
2 Xpr1 is an Atypical G-protein Coupled Receptor that Mediates
3 Xenotropic and Polytropic Murine Retrovirus Neurotoxicity
4
5 Andrew E. Vaughan,1 Ramon Mendoza,1 Ramona Aranda,1
6 Jean-Luc Battini,2 and A. Dusty Miller1*
7
8 Human Biology Division, Fred Hutchinson Cancer Research Center, 1100 Fairview
9 Avenue North, Seattle, WA 98109-1024, USA1; and Institut de Gntique Molculaire
10 de Montpellier, CNRS - UMR 5535, 1919 route de Mende, 34293 Montpellier, Cedex 5,
11 France2.
12
13 * Corresponding author. Mailing address: Fred Hutchinson Cancer Research Center,
14 1100 Fairview Avenue North, MS C2-023, Seattle, WA 98109-1024. Phone: (206) 667-
15 2890. E-mail:
dmiller@fhcrc.org.
16 Current address: Undergraduate Program, New Mexico State University.
17
18 Running title: Xpr1 is a GPCR that mediates retrovirus neurotoxicity.
19
20 Abstract word count: 226; Text word count: 5,563.
Copyright 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
J. Virol. doi:10.1128/JVI.06073-11
JVI Accepts, published online ahead of print on 16 November 2011
2
21 ABSTRACT
22
23 Xenotropic murine leukemia virus-related virus (XMRV) was first identified in human
24 prostate cancer tissue, and was later found in a high percentage of humans with chronic
25 fatigue syndrome (CFS). While exploring potential disease mechanisms, we found that
26 XMRV infection induced apoptosis in SY5Y human neuroblastoma cells, suggesting a
27 mechanism for the neuromuscular pathology seen in CFS. Several lines of evidence
28 show that the cell-entry receptor for XMRV, Xpr1, mediates this effect, and chemical
29 crosslinking studies show that Xpr1 is associated with the G? subunit of the G-protein
30 heterotrimer. Activation of adenylate cyclase rescued the cells from XMRV toxicity,
31 indicating that toxicity resulted from reduced G-protein-mediated cAMP signaling. Some
32 proteins with similarity to Xpr1 are involved in phosphate uptake into cells, but we found
33 no role of Xpr1 in phosphate uptake or its regulation. Our results indicate that Xpr1 is a
34 novel, atypical G-protein coupled receptor (GPCR) and that xenotropic or polytropic
35 retrovirus binding can disrupt the cAMP-mediated signaling function of Xpr1 leading to
36 apoptosis of infected cells. We show that this pathway also is responsible for the classic
37 toxicity of the polytropic mink cell focus-forming (MCF) retrovirus in mink cells. Although
38 it now seems clear that detection of XMRV in humans was the result of sample
39 contamination with a recombinant mouse virus, our findings may have relevance to
40 neurologic disease induced by MCF retroviruses in mice.
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41 INTRODUCTION
42
43 XMRV (xenotropic murine leukemia virus-related virus) was initially discovered in
44 human prostate cancer samples (36) and more recently, in the blood of a high
45 percentage of patients diagnosed with chronic fatigue syndrome (CFS) (20). Follow-up
46 studies found an even higher percentage of CFS patients harboring murine leukemia
47 virus (MLV) sequences in their peripheral blood cells (19), but these viral sequences
48 were closely related to known endogenous MLVs and not to the XMRV isolates, adding
49 confusion to the issue. Since these reports, many groups have been unable to confirm
50 the presence of XMRV in humans with prostate cancer or CFS (1, 14). Moreover, while
51 the specific sequence of XMRV initially appeared relatively unique to humans, a nearly
52 identical virus was found in a common prostate cancer cell line, 22Rv1 (13), and new
53 evidence indicates that this virus arose from recombination between two endogenous
54 mouse viruses during xenotransplantation of the cells in nude mice (27). Widespread
55 use of 22Rv1 cells and plasmid clones of XMRV suggest that detection of XMRV is due
56 to experimental contamination with such materials.
57 Because it initially appeared that XMRV was indeed a new human retrovirus, we
58 began studies to understand potential disease mechanisms. We first tested XMRV for
59 possible transforming activity that might explain a role for XMRV in prostate cancer, but
60 found no evidence that XMRV was acutely oncogenic (22). We next explored the
61 possibility that XMRV was neurotoxic, and that this might explain a role for XMRV in the
62 neuromuscular disease aspects of CFS. Indeed, several MLVs are known to have
63 neurologic and cytotoxic effects in animals and in cultured cells (32). Some cause
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64 paralytic motor neuron disease in mice, and the envelope (Env) proteins of these
65 viruses are often mechanistically involved. For example, CasBr-E MLV induces
66 spongiform neurodegeneration that is thought to involve interaction between the viral
67 Env protein and its cognate receptor mCAT-1 (17). Similarly, the Fr98 polytropic Friend
68 MLV induces astrogliosis in mice, and this neurovirulence is critically dependent on
69 specific amino acid residues in the Env protein (30, 31). We sought to determine if
70 XMRV had similar cytotoxic potential in vitro and to examine potential mechanisms
71 thereof.
72 Entry of xenotropic and polytropic retroviruses is mediated by the xenotropic and
73 polytropic cell-surface receptor Xpr1 (2, 35, 39), which has no documented function in
74 higher eukaryotes. While of unknown function, orthologs of Xpr1 are present in many
75 organisms, and include the yeast protein Syg1. In yeast, Syg1 is thought to be a
76 transmembrane signaling component that can respond to, or transduce signals through,
77 the G? subunit of the G-protein trimer (34). This is evidenced by the ability of a Syg1
78 truncation mutant, and to a lesser degree overexpression of wild type Syg1, to suppress
79 the lethality of G? deficiency.
80 G-protein signaling is important in a number of cellular processes, including
81 neurotransmission, metabolism, growth, and apoptosis (8). Based on its homology to
82 Syg1, we hypothesized that Xpr1 might play a similar role in G-protein signaling in
83 mammalian cells, and that xenotropic and polytropic MLV Env binding to Xpr1 might
84 disrupt its normal function. Here we show that Xpr1 does participate in G-protein
85 signaling, and that XMRV or polytropic retrovirus binding to Xpr1 in a human neuronal
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86 cell line, and polytropic retrovirus binding to Xpr1 in mink cells, induce apoptosis by
87 downregulation of cAMP-mediated G-protein signaling.
88
89 MATERIALS AND METHODS
90
91 Cell culture. Cells were grown in Dulbecco's modified Eagle medium (DMEM) with
92 10% fetal bovine serum (FBS) with the following exceptions. SY5Y cells (SH-SY5Y,
93 ATCC CRL-2266) (3) were grown in a 1:1 mixture of DMEM and Hams F12 medium
94 plus 15% FBS. CHO-K1 cells (11), which require proline for growth, were grown in
95 DMEM with 10% FBS and 1X non-essential amino acids (GIBCO). For study of
96 phosphate uptake and its regulation, CHO and 208F cells were grown in DMEM without
97 phosphate, 10% dialyzed FBS (to remove phosphate), and non-essential amino acids
98 for the CHO cells.
99 Viruses. XMRV was obtained from 22Rv1 cells (ATCC CRL-2505), which
100 constitutively produce XMRV (13). New Zealand black mouse (NZB) xenotropic
101 retrovirus was made by transfection of Mus dunni tail fibroblasts (MDTF cells) with the
102 NZB-9-1 plasmid (26). Because this plasmid contains a permuted copy of the NZB virus
103 that was cloned using the EcoRI site in the env gene, the plasmid was cut with EcoRI
104 and religated to generate intact copies of the virus prior to transfection. After
105 transfection, the cells were grown for 2 weeks to allow virus spread throughout the
106 culture. MCF 98D virus was produced from MDTF cells infected with a biological clone
107 of the virus (98D13) (gift from Bruce Chesebro). The LAPSN retroviral vector consists of
108 the human placental alkaline phosphatase cDNA cloned into the LXSN expression
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109 vector (24). All retroviruses were produced by feeding virus-producing confluent layers
110 of cells, by harvesting the culture medium 12 to 24 h later, and by filtering the medium
111 through 0.45 ?m-pore-size low-protein-binding filters to remove cells and debris. Viruses
112 were stored at ?70C.
113 Construction of hybrid amphotropic/MCF viruses. Plasmids containing an
114 amphotropic MLV provirus in which the env region was replaced with that of MCF 98D
115 were made by replacing the SphI to ClaI region in the amphotropic virus in the pAMS
116 plasmid (23) (GenBank accession AF010170) with the SphI to ClaI region of several
117 MCF 98D clones (GenBank accession AF133256) (kindly provided by Bruce Chesebro).
118 This region contains 640 bp of the pol gene that are upstream of the env coding region,
119 and all but 100 bp of the end of the env coding region. In the hybrid virus, the end of the
120 MCF env coding region downstream of the ClaI site is replaced with the nearly identical
121 region of the amphotropic env gene. Virus was made from these plasmids by
122 transfection of MDTF cells followed by passage of the cells for >2 weeks to allow virus
123 spread.
124 Analysis of apoptotic cells. SY5Y cells were plated in 12-well dishes at 5 104
125 cells per well, and the medium was replaced one day after plating. Two days after
126 plating, 100 ?l of virus (~106 infectious units) was added to each well with 4 ?g/ml
127 Polybrene to increase infection efficiency. The medium was replaced one day after
128 infection. Three days after infection, non-adherent cells were harvested and combined
129 with adherent cells harvested using trypsin, and the cells were analyzed for annexin V
130 binding by flow cytometry according to the manufacturers protocol (Invitrogen). In
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131 experiments utilizing forskolin, cells were pre-treated for 24 h and maintained in 50 ?M
132 forskolin.
133 Generation of retroviral vectors for expression of Xpr1 and Xpr1 truncation
134 mutants. Xpr1 cDNAs from human (hXpr1), Mus musculus (mXpr1) and Mus dunni
135 (mdXpr1) (GenBank accession numbers AF099082, AF198104, and AF198105,
136 respectively) were cloned into the LXSN retroviral expression vector (24). C-terminal
137 truncation mutants of human Xpr1, resulting in 200, 229, and 248 amino-acid proteins
138 were made by site-directed mutagenesis and were cloned into the LXSN vector. The
139 structures of the inserts were verified by DNA sequencing. Virus was made from the
140 vector plasmids by transient transfection of 293 cells, or by generation of stable
141 retrovirus packaging cell lines producing the vectors.
142 Cre-SEAP reporter assay. HEK 293 cells carrying the cAMP-responsive pCre-
143 SEAP reporter plasmid (a generous gift from Linda Buck) (18) were generated by
144 cotransfection of pCre-SEAP with a plasmid carrying the neo gene followed by selection
145 of the cells in G418. Clonal cell lines were isolated, and one that showed the highest
146 increase in SEAP production (12-fold) in response to overnight treatment with 15 ?M
147 forskolin, which increases cAMP levels by direct stimulation of adenylate cyclase, was
148 chosen for subsequent experiments.
149 For the SEAP assay, HEK 293/Cre-SEAP cells were seeded at 250,000 per well in
150 24-well plates. The following day, 0.5 ml of Xpr1-expressing or control retroviral vectors
151 were added to each well with 4 ?g/ml Polybrene and the cells were incubated overnight.
152 The following day, each plate was covered with plastic wrap and incubated at 68C for 2
153 h. Then 0.5 ml of 2 M diethanolamine, pH 10, containing 1.2 mM 4-methylumbelliferyl
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154 phosphate was added to each well and the cells were suspended by vigorous pipetting.
155 Fluorescence was measured every 30 min on a Fluoroskan Ascent with excitation at
156 360 nm and emission at 440 nm. Endpoint readings for each experiment were taken 3 h
157 after substrate addition.
158 Co-immunoprecipitation. NZB and AM-MLV SU- human IgG Fc hybrid proteins
159 were produced as previously described (2). The IgG Fc-tagged SU proteins were
160 concentrated using a Hi-Trap Protein G column to a final concentration of 10 ?g/ml by
161 following manufacturers protocol (GE Healthcare).
162 Nearly confluent SY5Y cells were harvested using phosphate-buffered saline (PBS)
163 without calcium or magnesium and containing 1 mM EDTA. Cells were washed and
164 resuspended in DMEM without serum, and 106 cells per assay were incubated with 2 ?g
165 of Env SU hybrid protein in a 0.5 ml volume for 1 h at 4C. Cells were washed once with
166 DMEM, once with PBS, were resuspended in 1 ml PBS containing 2 mM
167 dithiobis[succinimidyl propionate], and were incubated for 30 min at 25C. Next, 20 ?l of
168 1M Tris (pH 7) was added to each sample to stop the crosslinking reaction, and
169 samples were centrifuged at 800 g for 10 min. Samples were then resuspended in 0.5
170 ml lysis buffer (20 mM HEPES, 100 mM NaCl, 1 mM EDTA, 1% sodium dodecyl sulfate
171 [SDS] and protease inhibitors) and incubated for 1 h at 4C.
172 Meanwhile, Protein G Sepharose beads (GE Healthcare) were washed twice in lysis
173 buffer and then blocked at 4C with lysis buffer containing 1% bovine serum albumin
174 and 1% SDS for 1.5 h. Beads were then washed twice in lysis buffer, and were
175 resuspended the final time in lysis buffer without SDS. Crosslinked protein samples
176 were centrifuged at 15,000 g for 20 min to pellet insoluble material, and 50 ?l aliquots
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177 of each sample were saved for the input controls. 50 ?l protein G Sepharose beads
178 were added to the remainder of each sample and incubated overnight at 4C with
179 continuous rotation. The following day, the unbound lysate was discarded and beads
180 washed six times with lysis buffer plus 0.1% Tween 20, 0.1 % Triton X, and 0.5% SDS.
181 Samples were finally resuspended in 50 ?l lysis buffer containing 1% SDS. Loading
182 buffer containing ?-mercaptoethanol was added to each sample and incubated at 37C
183 for 30 min followed by 95C for 15 min. Samples were loaded onto a 15% SDS-PAGE
184 gel. After transfer, gels were probed with a pan G? antibody (sc-378, Santa Cruz
185 Biotechnology) followed by an anti-rabbit HRP secondary antibody (P0448, Dako) and
186 developed using ECL Plus reagents (Amersham).
187 Phosphate uptake assays. Chinese hamster ovary (CHO) and 208F rat fibroblasts
188 were transduced with Xpr1-expressing retroviral vectors and were selected in G418.
189 Cells were seeded at 2 to 4 105 cells per 3.5-cm-diameter dish in medium with various
190 concentrations of 32P-phosphate. Phosphate uptake was measured 1 day later as
191 previously described (12).
192 Statistical analysis. Statistical significance was determined by one-way analysis of
193 variance (ANOVA) and by using the Tukey method to compare pairs of means.
194
195 RESULTS
196
197 XMRV induces apoptosis in SY5Y neuroblastoma cells. To assay for
198 neurotoxicity that might be caused by XMRV, we infected various neural cell lines with
199 conditioned medium harvested from the 22Rv1 cell line, which produces high titer
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200 XMRV (13). Daoy meduloblastoma (10), U-251 glioma (4), and HEK 293 neuronal
201 lineage cells (33) appeared unaffected by XMRV infection, while SY5Y neuroblastoma
202 cells (3) displayed significant cell death starting two days after XMRV infection. By three
203 days after infection, measurement of apoptosis by annexin V staining revealed that
204 many more of the XMRV-infected cells had become apoptotic compared to mock205
infected cells or to cells infected with a related xenotropic retrovirus endogenous to NZB
206 mice, NZB-9-1 (26) (Fig. 1A and B). XIAP binds and inhibits several caspases in healthy
207 cells, and lower levels of XIAP are characteristic of cells undergoing apoptosis.
208 Consistent with the annexin V staining results, XMRV infection resulted in a lower level
209 of XIAP protein in SY5Y cells (Fig. 1C). The XIAP level of cells infected with the NZB
210 virus was between those of mock and XMRV infected cells, suggesting that the NZB
211 virus might also be somewhat toxic to SY5Y cells.
212 To address the possibility that 22Rv1 cells produced something in addition to XMRV
213 that was responsible for the SY5Y cell toxicity we observed, we produced XMRV from
214 the VP62 XMRV clone (7) and tested this virus for toxic effects on SY5Y cells. To obtain
215 virus with a high enough titer for this experiment, we transfected 293 cells with the VP62
216 clone or a plasmid expressing GFP as a control, exposed HT-1080 cells to medium
217 from the transfected 293 cells, passaged the HT-1080 cells for several weeks, and
218 exposed SY5Y cells to medium from the HT-1080 cells. Exposure of SY5Y cells to
219 medium made by transfection of the VP62 plasmid (VP62 TX) resulted in significantly
220 more apoptosis than that produced by medium made by transfection of the control GFP
221 plasmid (GFP TX) (Fig. 1D), showing that XMRV can indeed induce SY5Y cell
222 apoptosis. The titer of the VP62 virus measured by S+L? assay (7 105) was lower
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223 than that of XMRV produced by 22Rv1 cells (3 106), which correlates with the lower
224 toxicity of the VP62 virus compared to the 22Rv1 XMRV. Because of its higher titer, we
225 used the 22Rv1 XMRV virus for the experiments described below.
226 Expression of mouse Xpr1 protects SY5Y cells from XMRV-induced apoptosis.
227 The Env proteins of several retroviruses are known to affect normal receptor function.
228 For example, the Env protein of the 4070A amphotropic MLV can inhibit phosphate
229 uptake mediated by its receptor, Pit-2 (also called Ram-1, SLC20A2) (12). To address
230 the hypothesis that XMRV might cause cell death by affecting the function of its
231 receptor, SY5Y cells were transduced with a retroviral vector expressing Xpr1 cloned
232 from lab mice (mXpr1). While closely related to human Xpr1 (hXpr1), mXpr1 is unable to
233 mediate entry of xenotropic retroviruses, and presumably is unable to bind xenotropic
234 MLV Env protein. We found that SY5Y cells expressing mXpr1 exhibited much less
235 apoptosis following XMRV infection than did SY5Y cells transduced with the empty
236 vector or a vector expressing hXpr1 (Fig. 2). The mXpr1-transduced SY5Y cells
237 remained as susceptible to infection by XMRV as wild-type cells (XMRV-pseudotype
238 LAPSN vector titers of 4.1 104 and 3.9 104, respectively, N=2), indicating that the
239 SY5Y/mXpr1 cells still express endogenous hXpr1 at levels similar to those of SY5Y
240 cells. These results show that mXpr1 can protect SY5Y cells from XMRV induced
241 apoptosis, presumably by compensating for the loss of hXpr1 function that occurs
242 following binding of XMRV Env to hXpr1, and indicate that hXpr1 plays a key role in the
243 induction of apoptosis in SY5Y cells.
244 Xpr1 participates in G-protein-mediated cAMP signaling. Based on the similarity
245 of Xpr1 to Syg1, a yeast G-protein interactor involved in cAMP signaling, we measured
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246 cAMP levels after overexpression of human and mouse Xpr1 homologs in HEK 293
247 cells. To measure cAMP levels, the cells were transfected with the cAMP-responsive
248 reporter Cre-SEAP and were selected for stable reporter expression. These reporter
249 cells were then transduced with retroviral vectors that express Xpr1 genes at high
250 levels. Overexpression of both hXpr1 and mXpr1 resulted in significantly higher levels of
251 intracellular cAMP, as determined by SEAP expression from the reporter (Fig. 3A).
252 We reasoned that, like Syg1, Xpr1 might be inducing higher levels of cAMP by
253 binding the G? subunit of the G-protein complex, and liberating G? to activate adenylate
254 cyclase. We first attempted to use Xpr1 antibodies to co-immunoprecipitate proteins
255 bound to Xpr1, but none of the commercially available Xpr1 antibodies bound Xpr1.
256 Instead, we performed co-immunoprecipitation by using a hybrid protein consisting of
257 the NZB Env extracellular domain (SU) linked to a human IgG constant fragment, which
258 is known to specifically bind Xpr1 (2). NZB SU fusion protein bound in a complex to G?,
259 whereas an equivalent fusion protein containing an amphotropic Env SU, which binds
260 the unrelated retrovirus receptor Pit-2 (15), did not show significant binding (Fig. 3B).
261 These results show that Xpr1 associates with the G? component of the G-protein
262 heterotrimer.
263 XMRV infection decreases intracellular cAMP levels. We hypothesized that
264 XMRV toxicity in SY5Y cells is mediated by reduced cAMP levels resulting from XMRV
265 Env binding to Xpr1 and resultant inhibition of Xpr1 signaling. To test directly for
266 reduced cAMP levels, we utilized the Glosensor cAMP 22F system to analyze cAMP
267 levels in SY5Y cells. The Glosensor reporter consists of a luciferase enzyme that is
268 directly activated in proportion to the intracellular cAMP level. Infection of Glosensor13
269 expressing SY5Y cells with XMRV resulted in lower rates of luminescence increase,
270 indicative of lower levels of luciferase (Fig. 4A-B) and therefore lower intracellular cAMP
271 levels. Similarly, the polytropic retrovirus MCF 98D (5), which also binds Xpr1 but takes
272 longer to induce apoptosis in SY5Y cells (nine days after infection compared to three
273 days for XMRV) also reduced cAMP levels, although not to the same degree as XMRV.
274 Increased levels of cAMP protect cells from XMRV-induced apoptosis. To
275 further test the hypothesis that XMRV induces apoptosis by downregulating cAMP
276 levels in SY5Y cells, we treated cells with forskolin, a direct activator of adenylate
277 cyclase, and then infected the cells with XMRV. In contrast to untreated cells, forskolin
278 treatment significantly decreased the size of the apoptotic population of cells 3 days
279 after infection (Fig. 5). This indicates that increasing the cAMP level protects cells from
280 XMRV-induced apoptosis. Forskolin treatment also appeared to decrease the basal
281 level of apoptosis in mock-infected SY5Y cells (Fig. 5), although this trend was not
282 statistically significant (P > 0.05), suggesting that SY5Y cells constitutively maintain
283 levels of cAMP that are insufficient to prevent apoptosis.
284 Expression of carboxy terminal-deleted Xpr1 mutants protects SY5Y cells
285 against XMRV-induced apoptosis. C-terminal truncated forms of Syg1 in yeast are
286 known to suppress lethality of G? subunit deficiency by stimulating constitutive signaling
287 through G? (34). In fact, two of the truncated proteins (400 and 417 aa) were found to
288 be more active than the full-length Syg1. To test whether Xpr1 might have similar
289 properties, we made similar C-terminal truncation mutants of Xpr1 (200, 229 and 248
290 aa) by reference to the hydropathy plots of both proteins (Fig. 6A). Note that the lengths
291 of Xpr1 and Syg1 are quite different (696 versus 902 aa, respectively), with most of the
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292 difference in the N-terminal presumed cytoplasmic domain. The Xpr1 mutants were
293 cloned into a retroviral expression vector, and VSV-G-pseudotype vector preparations
294 were made for each of the mutants, for full-length hXpr1, and for the empty vector.
295 SY5Y cells were transduced with the vectors, were grown in G418 to select for
296 expression of the neo gene present in all of these vectors, and were tested for
297 resistance to XMRV-induced apoptosis. In contrast to SY5Y cells transduced with vector
298 alone, those expressing the Xpr1 truncations showed significantly reduced apoptotic
299 populations after XMRV infection (Fig. 6B). Basal levels of apoptosis appeared to be
300 reduced in cells expressing the truncation mutants (Fig. 6B), although this difference
301 was not statistically significant (P > 0.05). This effect is similar to the effect observed
302 following forskolin treatment (Fig. 5), and suggests that the Xpr1 truncation mutants can
303 increase constitutive cAMP levels and thereby decrease apoptosis.
304 Polytropic and xenotropic viruses can kill Mv1Lu mink lung cells. MCF
305 polytropic retroviruses are well known to be toxic to Mv1Lu mink lung epithelial cells in
306 tissue culture (9, 40). Indeed, this toxicity is the basis for the mink cell focus assay used
307 to quantitate MCF viruses (9). It has been proposed that this death is due to virus
308 superinfection and subsequent buildup of Env protein resulting in ER stress (41).
309 However, given the similarities between this phenomenon and XMRV toxicity to SY5Y
310 cells, we hypothesized that MCF might kill cells via a similar cAMP-mediated
311 mechanism.
312 We first tested for xenotropic retrovirus toxicity to mink cells in comparison to known
313 MCF toxicity. Cells infected with MCF 98D virus showed obvious toxicity within 3-5
314 days, with nearly complete cell death within the next week. XMRV showed limited
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315 toxicity that was not apparent until 18 days after infection. NZB virus infection showed
316 intermediate toxicity, with cell death apparent in 11-12 days. Note that we previously
317 reported that XMRV infection was not toxic to mink cells (22), but the virus used for
318 infection in that study was a mixture of XMRV with the LAPSN vector. The LAPSN
319 vector replicates more efficiently than XMRV (22), and this may explain why the virus
320 mixture showed no toxic effects. In contrast, pure XMRV virus reproducibly induced
321 toxicity in mink cells.
322 Forskolin treatment protects mink lung cells and SY5Y neuroblastoma cells
323 from polytropic retrovirus-induced death. To determine whether cAMP levels play a
324 role in polytropic retrovirus toxicity in mink cells, mink cells were infected with MCF 98D
325 virus, and when toxicity was apparent on day 3, the cells were trypsinized and replated
326 with or without 25 ?M forskolin, which directly stimulates cAMP production. The MCF327
infected mink cells treated with forskolin resumed their growth, while those without
328 continued to exhibit toxicity, and by day 10 after infection were mostly dead (Fig. 7). The
329 forskolin-treated MCF-infected cells continued to grow, although not as quickly as
330 uninfected mink cells, until the experiment was terminated on day 24. On day 17, when
331 these cells were trypsinized and reseeded, some cells were cultured in parallel without
332 forskolin. The cells grown with and without forskolin appeared to grow at a similar rate
333 until day 21, after which most of the cells grown without forskolin died (Fig. 7). These
334 results show that forskolin, a direct activator of adenylate cyclase, can reverse MCF
335 retrovirus-induced toxicity in mink cells.
336 We performed additional experiments to test for a role of cAMP in SY5Y killing by
337 the MCF 98D polytropic retrovirus. Although it took longer for the MCF 98D virus to kill
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338 SY5Y cells than did XMRV (9 versus 2 days, respectively), killing by MCF 98D was
339 dramatic. At day 12, when most of the MCF infected cells were dead, we trypsinized
340 and replated the cells with or without 50 ?M forskolin, after which the forskolin treated
341 cells recovered and grew well until the experiment was terminated 1 week later, while
342 the untreated cells continued to die. Overall, these results indicate that decreased
343 cAMP levels mediate the toxicity of both XMRV and the MCF 98D polytropic retrovirus
344 in SY5Y cells.
345 Killing of mink cells by MCF 98D virus is dependent on the MCF env gene. To
346 test the hypothesis that MCF virus toxicity was mediated by Env interaction with Xpr1,
347 we generated hybrid viruses comprised of a non-cytotoxic amphotropic retrovirus into
348 which several cDNA clones of the env coding region of MCF 98D virus were inserted.
349 We produced virus from plasmids containing these clones by transfection of the
350 plasmids into MDTF cells and by growing the cells for >2 weeks to allow virus spread.
351 Mink cells were exposed to medium harvested from MDTF cells, MDTF cells
352 producing intact MCF virus or the ampho/MCF hybrid viruses, medium from NIH 3T3
353 cells producing amphotropic virus following transfection with the pAMS plasmid, or to
354 fresh culture medium only. The cells in each dish were trypsinized and replated at a
355 1:10 dilution every time they reached confluence. By day 3 after exposure, toxicity was
356 evident in mink cells exposed to two of the ampho/MCF hybrid viruses and the intact
357 MCF virus (two experiments). In contrast, during a month of observation, no difference
358 in growth rate or appearance of cells exposed to medium from untransfected MDTF
359 cells, fresh culture medium, medium containing the amphotropic virus, or medium from
360 one of the three ampho/MCF clones tested. We assume that the lack of cytotoxicity
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361 observed for one of the ampho/MCF clones was due to a disabling mutation, but have
362 not confirmed this. The same pattern of toxicity for the three ampho/MCF clones was
363 observed after infection of SY5Y cells, with toxicity due to two of the clones becoming
364 apparent on day 8 after virus exposure. These results show that the cytotoxic effects of
365 the MCF virus are linked to the MCF env gene, and are consistent with a role of Xpr1 in
366 MCF cytotoxicity.
367 Xpr1 has little or no role in phosphate uptake or its regulation. Inorganic
368 phosphate is a limiting nutrient in the environment, and cells from most organisms
369 express multipass membrane-spanning proteins that actively concentrate phosphate
370 within cells. The hydrophilic amino-terminal region of Xpr1 shows similarity to proteins
371 involved in the regulation of phosphate uptake, e.g., yeast PHO81, neurospora PHO85
372 and NUC-2, and rice and arabidopsis SPX proteins (16, 28, 38). To test for a role for
373 Xpr1 in phosphate uptake, we measured the kinetics of radiolabeled inorganic
374 phosphate (Pi) uptake by 208F rat fibroblasts and Chinese hamster ovary (CHO) cells
375 transduced with retroviral vectors that express hXpr1 or the Xpr1 ortholog from Mus
376 dunni wild mice (mdXpr1). A retroviral vector expressing Pit2, the cellular receptor for
377 amphotropic MLV and a known sodium-dependent phosphate symporter (12), was used
378 as a positive control, and the empty vector LXSN was used as a negative control. Pi
379 uptake by cells expressing hXpr1 was no different from that of cells transduced with the
380 empty vector, while Pi uptake by cells expressing rat Pit2 (rPit2) (25) was dramatically
381 increased (Fig. 8A-B). There was a small but reproducible decrease in phosphate
382 uptake by 208F and CHO cells expressing mdXpr1. These experiments indicate that
18
383 hXpr1 and mdXpr1 are not primary phosphate transporters, as is rPit2, but suggest the
384 possibility that mdXpr1 has some ability to downregulate Pi uptake.
385 To further investigate possible regulatory activity of mdXpr1, we measured the effect
386 of mdXpr1 expression on the response of CHO cells to various concentrations of
387 external Pi. We found that CHO cells exposed to culture medium without phosphate up388
regulate their endogenous phosphate uptake activity, such that the Vmax of phosphate
389 uptake was increased by 2-fold after 4 h, and by 5-fold after 15 h, showing that CHO
390 cells actively regulate phosphate uptake. The response of CHO cells to an overnight
391 incubation with 0 or 10 nM Pi was similar whether they expressed mdXpr1 or not, and
392 the response to a 15 h exposure to 100 nM Pi was identical (Fig. 8C). There was still a
393 small difference in Vmax between the populations of CHO cells cultured in 1 mM Pi, the
394 concentration of Pi present in standard culture medium, consistent with our previous
395 results (Fig. 8A-B).
396 To confirm that the Xpr1 orthologs were being expressed in CHO cells, and that the
397 lack of alteration in phosphate transport activity that we observed was not simply
398 because the Xpr1 orthologs were not being expressed, we tested for Xpr1 function as a
399 retrovirus receptor in CHO cells (Table 1). CHO cells are naturally resistant to
400 xenotropic, polytropic, and amphotropic retrovirus infection, as confirmed in cells
401 transduced with the empty vector (Table 1, bottom row). Transfer of hXpr1 or mdXpr1
402 genes into the cells rendered them susceptible to xenotropic and polytropic, but not
403 amphotropic retroviruses, as expected. Human cells are somewhat less infectable by
404 polytropic retroviruses, and this is reflected in the data. CHO cells transduced with the
405 mXpr1 gene were susceptible to polytropic but not to xenotropic or amphotropic
19
406 retrovirus infection, again as expected. These results show that hXpr1, mXpr1 and
407 mdXpr1 are expressed in CHO cells transduced with the respective Xpr1 genes.
408 Overall, our results indicate a limited if any role of Xpr1 in phosphate uptake or its
409 regulation.
410
411 DISCUSSION
412
413 Xpr1 has been well characterized as a cellular receptor for polytropic and xenotropic
414 MLVs, but its physiological function, aside from that predicted by its homology to the
415 yeast signaling protein Syg1, has not been characterized. Xpr1 is a highly conserved
416 protein with orthologs in animals, plants, and unicellular organisms, indicating its
417 importance in basic cellular functions. This is underscored by the finding that Xpr1
418 deletion in mice is embryonic lethal (personal communication, Lexicon
419 Pharmaceuticals), indicating an important role for Xpr1 in mammals.
420 Here we demonstrate that hXpr1 regulates cAMP in a manner analogous to known
421 GPCRs, and that it interacts with the G? subunit of the large G protein complex. While a
422 physiologic ligand for Xpr1 is unknown, we can force signaling through the Xpr1
423 pathway by Xpr1 overexpression. Like known G? stimulatory GPCRs, Xpr1 activation
424 increases intracellular levels of cAMP, which acts as a second messenger and induces
425 downstream gene expression of CREB-responsive genes. However, unlike all other
426 described GPCRs, Xpr1 contains eight putative transmembrane domains, as opposed
427 to the canonical seven, and Xpr1 shows no sequence similarity to known GPCRs.
428 Despite some similarity of Xpr1 to known phosphate transporters and phosphate
20
429 transport regulators in yeast, neurospora, rice, and arabidopsis, we were unable to
430 demonstrate a role for Xpr1 in phosphate uptake.
431 Interestingly, expression of the 200, 229 and 248 aa C-terminal truncations of Xpr1,
432 which lack most or all of the Xpr1 transmembrane domains, induced constitutive
433 signaling. We hypothesize this to be due to strong binding of G? by these truncated
434 Xpr1 proteins, with release of G? into the cytoplasm, constitutive activation of adenylate
435 cyclase, and a resulting increase in cAMP levels. These results parallel findings with the
436 400 and 417 aa C-terminal truncated Syg1 proteins in yeast (34), and indicate that the
437 N termini of Xpr1 and Syg1 have similar function despite having markedly different
438 lengths and only 29% amino acid similarity (if multiple gaps in the alignment are
439 ignored) or 17% similarity (if gaps are included).
440 We found that several retroviruses that utilize Xpr1 as a receptor for cell entry could
441 inhibit Xpr1-mediated cAMP production resulting in apoptosis and death of some cell
442 types. Interestingly, XMRV showed the most rapid toxic effect in SY5Y cells, while the
443 MCF 98D virus showed the most rapid toxic effect in Mv1Lu mink lung cells. NZB virus
444 had an intermediate effect in mink cells and had lower if any toxicity than XMRV in
445 SY5Ycells. All of these viruses can use the Xpr1 orthologs expressed in SY5Y and mink
446 cells for cell entry. Presumably, the differential toxicity of these viruses is due largely to
447 differences in their interaction with Xpr1 orthologs from different species. However,
448 beyond initial receptor binding by Env, we do not know the mechanism of toxicity, which
449 may involve downregulation of Xpr1 signaling, or degradation of Xpr1/Env complexes
450 during protein synthesis or following internalization from the cell surface.
21
451 While it seems clear now that XMRV is not associated with CFS, our study provides
452 unique insight into a potential role for Xpr1 in neural biology. A role for deregulated
453 cAMP signaling has clear precedents in other disease models and developmental
454 observations. For example, mouse embryos lacking CREB, a major downstream
455 transducer of G-protein signaling, exhibit excess apoptosis and degeneration of sensory
456 and sympathetic neurons, and CREB knockout is ultimately embryonic lethal (21). Our
457 studies show that CREB is activated by Xpr1 signaling because CREB is the
458 transcription factor that drives the SEAP expression in the assay we employed to
459 measure cAMP levels. In addition, cAMP-modulating drugs have been shown to reverse
460 neurodegeneration in cultured hippocampal neurons treated with amyloid ?-peptide, a
461 model of Alzheimers disease (37). Moreover, mice infected with multiple neuropathic
462 polytropic MLVs display increased expression of proinflammatory cytokines and
463 chemokines, and neurodegenerative diseases such as Alzheimers and multiple
464 sclerosis induce similar cytokine and chemokine expression (29).
465 Eurexpress Transcriptome Atlas data indicates that brain tissues show the highest
466 Xpr1 expression levels in the mouse (6), lending support to the hypothesis that Xpr1 is
467 involved in normal brain function. Notably, neuropathology can be induced in mice
468 following infection with various retroviruses, and some studies have shown that
469 polytropic retroviruses are neuropathic while related ecotropic retroviruses that do not
470 use Xpr1 as a receptor are not (29). However, neuropathology is also found in mice
471 following infection by some ecotropic retroviruses, such as CasBr-E MLV, that do not
472 use Xpr1 as a receptor (32). Infection of mice with ecotropic retroviruses frequently
473 results in recombination events leading to production of polytropic retroviruses (32), and
22
474 it would be interesting to determine if these recombinant viruses play a role in
475 neurologic disease induced by ecotropic retroviruses.
476 Our findings demonstrate the critical role Xpr1 plays in mediating both xenotropic
477 and polytropic retrovirus pathology as well as further elucidating the function of Xpr1 in
478 normal cells. However, there remain many questions regarding the normal physiological
479 role of Xpr1 in animals, including what extracellular ligands might modulate Xpr1
480 activity. While our data indicate that Xpr1 provides a pro-survival signal to the cell, a
481 more specific role for Xpr1 in signal transduction remains to be determined. It will be
482 important to understand whether Xpr1 acts in a fashion analogous to other GPCRs and
483 transmits a signal for a particular cellular process, or whether it regulates GPCR and
484 cAMP signaling in a more global fashion.
485
486 ACKNOWLEDGEMENTS
487
488 This work was supported by the Fred Hutchinson Cancer Research Center (A.E.V.
489 and A.D.M.), Pilot Project funding from the Core Center of Excellence in Hematology
490 grant DK56465 (A.D.M.), training grant T32 CA080416 from the National Cancer
491 Institute (R.M.), and by funding from a summer undergraduate internship program
492 (R.A.). J.L.B. is currently supported by INSERM, the Association Franaise contre les
493 Myopathies, and the Philippe Foundation.
494 We thank Linda Buck for providing the Cre-SEAP reporter vector, Bruce Chesebro
495 for MCF 98D retrovirus and plasmid clones, and shared resources at the Fred
23
496 Hutchinson Cancer Research Center for assistance with flow cytometry and DNA
497 sequencing.
24
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640
641
642
31
643 Table 1. Homo sapiens, Mus musculus and Mus dunni Xpr1 clones function as
644 retrovirus receptors when expressed in CHO cellsa
645
646 Vector titer for LAPSN pseudotype (virus strain):
647 Xpr1 Xenotropic Polytropic Amphotropic
648 ortholog (NZB) (MCF 98D) (4070A)
649 hXpr1 4 106 3 104 <1
650 mXpr1 40 5 107 <1
651 mdXpr1 2 106 6 107 <1
652 None 30 <1 <1
653
654 a Human (h), Mus musculus (m) and Mus dunni (md) Xpr1 cDNAs were cloned into
655 the LXSN retroviral expression vector, retroviruses were made from the vector plasmids
656 by transfection of 293 cells with each vector and Moloney MLV gag-pol and VSV-G env
657 genes, and CHO cells were exposed to the viruses and grown in G418 to select for the
658 neo gene carried by the LXSN vector. To test for the ability of xenotropic, polytropic and
659 amphotropic retroviruses to infect the CHO cells carrying the different xpr1 genes, the
660 cells were exposed to virus harvested from MDTF cells transduced with the LAPSN
661 vector and the indicated replication-competent xenotropic or polytropic retroviruses, or
662 to LAPSN vector produced by PA317 retrovirus packaging cells that express the 4070A
663 amphotropic retrovirus Env protein. Two days later, the CHO cells were stained for
664 alkaline phosphatase (expressed by the LAPSN vector) to determine the vector titer.
665 Results are means of two to four experiments.
666
32
667 FIGURE LEGENDS
668
669 Fig. 1. XMRV induces apoptosis in SY5Y cells. (A) Representative FACS histogram
670 showing annexin V staining of SY5Y cells three days after infection with the indicated
671 retroviruses. The black bar represents the gate used to determine the percentage of
672 apoptotic cells. (B) Quantitation of apoptosis measured by FACS three days after
673 infection. Results are means S.D. from 7 experiments. (C) XIAP expression three
674 days after retrovirus infection of SY5Y cells was determined by western blotting using
675 equal amounts of total cell protein (Bradford assay) and XIAP antibody cat. no. 610762
676 (BD Biosciences). (D) Quantitation of apoptosis by FACS three days after exposure of
677 SY5Y cells to medium made by transfection of the VP62 XMRV plasmid (VP62 TX) or a
678 control plasmid expressing GFP (GFP-TX), or medium containing XMRV from 22Rv1
679 cells. Results are means S.D. from 2 experiments.
680
681 Fig. 2. Expression of mXpr1 protects SY5Y cells from XMRV-induced apoptosis. SY5Y
682 cells transduced with retroviral vectors encoding the indicated Xpr1 proteins or the
683 empty vector were exposed to medium containing the indicated retroviruses or fresh
684 medium only (mock) and the percentages of apoptotic cells were measured 3 d later.
685 Results are means SD of two (empty vector and human Xpr1) or three (mouse Xpr1)
686 experiments.
687
688 Fig. 3. Overexpression of mXpr1 or hXpr1 increases cAMP levels, and hXpr1 is
689 associated with the G? G-protein subunit. (A) cAMP levels in HEK 293 cells were
33
690 measured using the Cre-SEAP reporter 24 h post-transduction with vectors encoding
691 mXpr1, hXpr1 or with the empty vector, as indicated. Results are means SD of three
692 independent experiments. (B) SY5Y cells were incubated with IgG-Fc tagged NZB or
693 amphotropic Env SU proteins, or no protein (mock). Total proteins were crosslinked,
694 coimmunoprecipitated using anti IgG-Fc antibody, and analyzed for the presence of the
695 G? protein by western blotting with a pan G? antibody. Proteins present in the cell
696 lysate made after protein crosslinking (input) and after co-immunoprecipitation (co-ip)
697 are shown. Lower bands in the co-ip lanes are due to dissociated protein G from the
698 Sepharose beads. Input lanes indicate equivalent amounts of G? in the cell lysates prior
699 to co-immunoprecipitation.
700
701 Fig. 4. XMRV and MCF 98D virus infection of SY5Y cells decreases intracellular cAMP.
702 (A) SY5Y cells containing the Glosensor cAMP 22F reporter construct were infected
703 with virus and assayed for luciferase activity three days later. Luminescence as a
704 function of time is plotted following addition of luciferin to the cells. Each line represents
705 the average of three wells in a 96-well plate. (B) Fold changes in rate of luminescence
706 gain relative to mock-infected cells are shown. Results are means SD of two
707 experiments.
708
709 Fig. 5. Forskolin protects SY5Y cells from XMRV toxicity. SY5Y cells were cultured with
710 or without 50 ?M forskolin for 24 h prior to infection and for 72 h after infection with the
711 indicated viruses. Results are means S.D. of seven experiments. Note that the data
712 for untreated (no forskolin) cells are also shown in Fig. 1B. The statistical analysis
34
713 shown here was performed by ANOVA using all data from untreated and treated cells,
714 while that for Fig.1B only used only the untreated cell data.
715
716 Fig. 6. Truncation mutants of Xpr1 provide protection against XMRV-induced
717 apoptosis. (A) Hydropathy plots of the yeast (S. cerevisiae) Syg1 and human Xpr1
718 proteins are shown. The Xpr1 plot was shifted to the right to align the first putative
719 transmembrane domain of each protein. The locations of the last amino acid of the C720
terminal truncation mutations are shown. The 400 and 417 aa Syg1 truncations were
721 found to be fully active in yeast (34). (B) SY5Y cells transduced with retroviral vectors
722 encoding the indicated hXpr1 truncation mutants, or the empty vector, were cultured
723 and infected with the indicated retroviruses. Three days after infection, the percentages
724 of apoptotic cells were measured by annexin V staining and flow cytometry. Results are
725 means SD of three independent experiments.
726
727 Fig. 7. Forskolin protects mink cells from polytropic (MCF) virus-induced death.
728 20%-confluent Mv1Lu mink lung epithelial cells (ATCC CCL-64) were exposed to ~107
729 infectious units of MCF 98D or no virus in the presence of 4 ?g/ml Polybrene. Cells
730 were trypsinized and seeded at 1:10 and 1:40 dilutions every time they reached
731 confluence. Cells were visually monitored daily for the presence of any cytopathic
732 effects and cell death. Cells were treated with 25 ?M forskolin (FSK) as indicated. Cell
733 numbers were normalized to the starting cell number, and were approximated based on
734 the time it took to reach confluence again after a 1:10 or 1:40 split. Results using two
35
735 different preparations of MCF virus harvested from MDTF cells infected with MCF 98D
736 virus were the same.
737
738 Fig. 8. Expression of human or Mus dunni Xpr1 does not result in increased phosphate
739 uptake or significant alteration in regulation of uptake. (A) Retroviral vectors encoding
740 human (hXpr1) or Mus dunni Xpr1 (mdXpr1) were expressed in 208F rat cells that were
741 used for phosphate uptake analysis. Eadie-Hofstee plots of phosphate uptake velocity
742 (V) versus the velocity/phosphate concentration ratio (V/[Pi]) are shown. The Y-axis
743 regression line intercept gives the Vmax for phosphate uptake, and the slope is equal to
744 -Km. (B) The same phosphate uptake experiment described in (A) was performed using
745 CHO cells. (C) 208F cells transduced with the mdXpr1-expressing vector or the empty
746 vector LXSN were incubated in the indicated concentrations of phosphate overnight and
747 phosphate uptake was measured.
Eco
