www.sciencemag.org/cgi/content/full/science.1212182/dc1 Supporting Online Material for Partial Retraction to Detection of an Infectious Retrovirus, XMRV, in Blood Cells of Patients with Chronic Fatigue Syndrome Vincent C. Lombardi, Francis W. Ruscetti, Jaydip Das Gupta, Max A. Pfost, Kathryn S. Hagen, Daniel L. Peterson, Sandra K. Ruscetti, Rachel K. Bagni, Cari Petrow-Sadowski, Bert Gold, Michael Dean, Robert H. Silverman,* Judy A. Mikovits *To whom correspondence should be addressed. E-mail: silverr@ccf.org This PDF file includes: Materials and Methods SOM Text Figs. S1 to S4 Tables S1 and S2 References Posted 22 September 2011 DOI: 10.1126/science.1212182
Presence of XMRV Plasmid in Samples of Chronic Fatigue Syndrome Patient DNA Background In 2009, Lombardi et al.(1) reported an association between the retrovirus xenotropic murine leukemia virus-related virus (XMRV) and chronic fatigue syndrome (CFS), a debilitating disease of unknown etiology. The evidence included detection of XMRV DNA by PCR, culturing infectious XMRV from plasma and blood cells, electron microscopy of viral particles, and presence of circulating antibody reactive against a murine leukemia virus (MLV) envelope protein. However, with the exception of a single report of different polytropic and modified-polytropic MLV sequences in CFS blood (2), no other research team has published a confirmatory report. Instead, there have been at least 16 failed attempts to detect XMRV in CFS patients [e.g. (3, 4)]. To arrive at the truth about the association, or lack thereof, between XMRV and CFS, it is important to investigate the reasons for these discrepancies. Materials and Methods In early 2009 we received PBMC DNA samples from CFS patients and healthy controls from the Whittemore Peterson Institute (WPI), Reno, Nevada. The PBMC DNA samples were taken directly to a clean room upon arrival and stored in a -20 o C freezer in the same room. Precautions were taken to minimize the possibility of crosscontamination of the human samples with laboratory sources of XMRV DNA. In particular, neither plasmid XMRV VP62/pcDNA3.1(-) nor XMRV PCR products were ever taken into the clean room. Also, new pipetmans (Gilson) were purchased for exclusive use in the clean room and were never used elsewhere. At the entrance to the clean room there is a sticky pad on the floor and lab personnel must change lab coats upon entering and exiting the clean room. The clean room is locked when not in use. The PCR reaction mixtures that contained PBMC DNA were pipetted in an AirClean 600 PCR Work Station (ISC Bio Express) in the clean room. The PCR Work Station was purchased for use in the clean room and never used elsewhere. The single-round PCR on human DNA samples was performed in a BioRad PCR thermocycler, used exclusively for that purpose, in a separate room from the clean room. The PCR on the plasmid XMRV VP62/pcDNA3.1(-) was performed in yet another room in a different PCR thermocycler from the one used on patient DNA samples. The samples that were re-examined in this report were previously used to produce Figures 1 and S2 and Table S1 of Lombardi et al. (1). The CFS and healthy control PBMC DNA samples were treated identically. To detect DNA sequences of the XMRV genome and of the parent cloning vector, pcdna3.1(-) (Invitrogen), specific PCR primers were designed (Table 1). Single-round PCR and sequencing of the PCR products from PBMC DNA and from plasmid DNA were performed as described previously(1). PCR products were obtained from the XMRV env gene, the neomycin (neo) gene in pcdna3.1(-), and a junction consisting of the CMV promoter in pcdna3.1 linked to 5 - XMRV sequences (Fig. 1). In addition, PCR for the glyceraldehyde-3-phosphate dehydrogenase gene, GAPDH, was performed to confirm the presence of PBMC DNA in all of the samples. Results and Discussion Gel electrophoresis showed that 6 of 15 CFS patient DNA samples (WPI-1104, WPI- 1106, WPI-1115, WPI-1125, WPI-1178, and WPI-1179) amplified a region of XMRV env (Fig. 2A). In contrast, all 17 of the healthy control samples were negative for XMRV env (Fig. 2B). However, all of the CFS samples that were positive for XMRV env DNA were also positive for neo DNA, a part of XMRV VP62 plasmid (Fig. 1), whereas none of the 1
control samples were positive for neo DNA. To verify the presence of XMRV VP62 plasmid, a junction region between the CMV promoter in the plasmid and the 5 -region of XMRV was amplified (Fig. 1). PCR on the same 6 CFS DNA samples that were positive for env and neo DNA also amplified the CMV promoter in the plasmid fused to the 5 - region of the XMRV genome (containing R, U5 and part of the glyco-gag coding sequence), while none of the control samples were positive for the plasmid/5 -XMRV junction DNA (Fig. 2). Therefore, 6 of 15 CFS DNA samples were positive for all three PCR products (XMRV env, neo, and plasmid/5 -XMRV junction), while all of the control samples were negative for all three amplicons. The identity of PCR products for XMRV env, neo, and plasmid/xmrv junction were confirmed by DNA sequencing (Table 2). In addition, we amplified and sequence-verified the junction sequence from a seventh CFS DNA sample, WPI-1124 (Table 2). All seven of the XMRV DNA positive CFS samples from Figure 1 of the original study(1), were positive here for the XMRV VP62 plasmid junction. The sequences of the PCR products of the junction and neo obtained from representative DNA samples, WPI-1178 and WPI-1115, respectively, are shown (Figs. 3 & 4). Sequencing of the band near the size of the neo PCR product from sample WPI- 1174 showed that it was not neo but rather non-specific amplification. There were no examples of XMRV env DNA in the absence of plasmid sequences (Fig. 2). Two of the sequence-confirmed junction sequences (WPI-1104 and WPI-1178) were from same samples used to generate Table S1 (sequences of XMRV genomes) and Figure S2 (the phylogenetic tree) (1). It appears likely, therefore, that these XMRV sequences originated not from the patients but rather from the XMRV VP62 plasmid. We conclude the results in Figures 1 and S2 and Table S1 of Lombardi et al.(1) were spurious due to contamination with XMRV plasmid DNA. References 1. V. C. Lombardi et al., Science 326, 585 (Oct 23, 2009). 2. S. C. Lo et al., Proc Natl Acad Sci U S A 107, 15874 (Sep 7, 2010). 3. K. Knox et al., Science, (Jun 2, 2011). 4. C. H. Shin et al., J Virol, (May 4, 2011). 2
Table 1. PCR Primers Used In Single-Round PCR. neo 8F 5'-ACA AGA TGG ATT GCA CGC AGG TTC-3' neo 416R 5'-ATG TTT CGC TTG GTG GTC GAA TGG-3' CMV 385F 5 -XMRV 528R env 5922F env 6273R gapdh F gapdh R 5'-TGA TGC GGT TTT GGC AGT ACA TCA ATG-3' 5'-GCG TAA AAC CGA AAG CAA AAA TTC AGA CG-3' 5'-GCT AAT GCT ACC TCC CTC CTG G-3' 5'-GGA GCC CAC TGA GGA ATC AAA ACA GG-3' 5 -GAA GGT GAA GGT CGG AGT C-3 5 -GAA GAT GGT GAT GGG ATT TC-3 Table 2. PCR Products Sequenced. CFS Patient Code Region sequenced Results WPI 1104 CMV Pr/5 -XMRV Sequence Confirmed WPI 1106 CMV Pr/5 -XMRV Sequence Confirmed WPI 1115 CMV Pr/5 -XMRV Sequence Confirmed WPI 1124* CMV Pr/5 -XMRV Sequence Confirmed WPI 1125* CMV Pr/5 -XMRV Sequence Confirmed WPI 1178 CMV Pr/5 -XMRV Sequence Confirmed WPI 1179 CMV Pr/5 -XMRV Sequence Confirmed WPI 1104 neo Sequence Confirmed WPI 1115 neo Sequence Confirmed WPI-1174 neo Non-specific product WPI 1178 neo Sequence Confirmed WPI 1104 env Sequence Confirmed WPI 1106 env Sequence Confirmed WPI 1178 env Sequence Confirmed *DNA from cultured PBMC were used. 3
Figure 1. Map of plasmid XMRV VP62/pcDNA3.1 aligned to positions of PCR primers and products. PCR products were: CMV Pr/5 -XMRV (CMV 385F-XMRV 528R, 874 nt); env 5922F-6373R, and neo 8F-416R. Regions of the XMRV genome shown include: R, repeat; U5 and U3 regions of the long terminal repeat, gag-pro-pol gene, and the env gene. Plasmid pcdna3.1(-) regions shown include: CMV Pr, CMV promoter; BGH pa, BGH polyadenylation sequence; f1 ori, f1 origin; SV40 ori, SV40 early promoter and origin; neomycin resistance gene; SV40 pa, SV40 early polyadenylation signal; puc ori, puc origin; and the ampicillin resistance gene. 4
Figure 2. XMRV and plasmid sequences in PBMC DNA from CFS patients. Singleround PCR results for XMRV env, neo, plasmid CMV promoter (Pr) joined to 5 -XMRV DNA (CMV Pr/5 -XMRV), and gapdh sequences in PBMCs of (A) CFS patients and (B) healthy controls. The positions of the amplicons are indicated and DNA markers (ladder) are shown. The positive control was plasmid XMRV VP62/pcDNA3.1 (pxmrv). The DNA from sample WPI-1125 only was from cultured PBMC. *Positive/Total: number of PCR products obtained for env, neo, and CMV Pr/5 -XMRV divided by three. 5
WPI-1178 CMV 385F 1 TGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTC CMV ------------------------------------------------------------ 61 CAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACT ------------------------------------------------------------ 121 TTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGT ------------------------------------------------------------ 181 GGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTA ------------------------------------------------------------ 241 TCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACGGGCCCT ----------------------------------------------------> MCS ------> 301 CTAGACTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTTAGTCATCCGA ------------------> XMRV R --------> 361 TAGACTGAGTCGCCCGGGTACCCGTGTTCCCAATAAAGCCTTTTGCTGTTTGCATCCGAA -----------------------------------------------------> U5 ------ 421 GCGTGGCCTCGCTGTTCCTTGGGAGGGTCTCCTCAGAGTGATTGACTACCCAGCTCGGGG ------------------------------------------------------------ 481 GTCTTTCATTTGGGGGCTCGTCCGGGATTCGGAGACCCCCGCCCAGGGACCACCGACCCA ---------> trna-pro PBS -----------------> 541 CCGTCGGGAGGTAAGCCGGCCGGCGATCGTTTTGTCTTTGTCTCTGTCTTTGTGCGTGTG splice donor------> 601 TGTGTGTGCCGGCATCTAATCCTCGCGCCTGCGTCTGAATCTGTACTAGTTAGCTAACTA 661 GATCTGTATCTGGCGGTTCCGCGGAAGAACTGACGAGTTCGTATTCCCGGCCGCAGCCCT glyco-gag start --> 721 GGGAGACGTCCCAGCGGCCTCGGGGGCCCGTTTTGTGGCCCATTCTGTATCAGTTAACCT 781 ACCCGAGTCGGACTTTTTGGAGTGGCTTTGTTGGGGGACGAGAGACAGAGACACTTCCCG 841 CCCCCGTCTGAATTTTTGCTTTCGGTTTTACGC <---------------------------- XMRV 528R Figure 3. Sequence of the CMV Pr/5 -XMRV PCR product from CFS PBMC DNA sample WPI-1178. Oligonucleotide primer or primer binding sites are shown in bold and italic. MCS, multiple cloning site; trna-pro PBS, prolyl trna primer binding site. 6
WPI-1115 neo 8F 1 ACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGA 61 CTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGG 121 GCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGA 181 GGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGT 241 TGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCT 301 GTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCT 361 GCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACAT neo 416R Figure 4. Sequence of the neo 8F-416R PCR product from CFS PBMC DNA sample WPI-1115. Jaydip Das Gupta and Robert H. Silverman Department of Cancer Biology Cleveland Clinic, Cleveland, OH 44195 7