Discovery and falsification of XMRV 21 Linkage Rnasel Hpc1

XMRV isolation was not a sudden or isolated finding, but it rather stemmed out of approximately twenty years of research by several groups, with a leading role by the group of R. Silverman [3] [4]. This work, as well, has even older roots, since it was initiated by decipher ing the antiviral response triggered by Interferon (IFN). Robert Silverman's work was pioneering and seminal in this effort: together with Ian Kerr, he clarified the Interferon (IFN) response to viral infection, initially by characterizing the 5'-triphosporylated, 2',5'-linked oli-goadenylates or 2-5A, a second messenger in the IFN response and its synthesizing enzyme (oligo-2',5'-A synthethase, or OAS) and finally discovering that 2-5A is the activator of an endogenous RNase activity, called RNase L [5] [3]. This is ubiquitously distributed but inactive inside cells, but it becomes strongly activated by binding 2-5A. By using radiolabelled 2-5A as probe, Silverman was able to identify and clone the gene RNASEL and to map later its location on chromosome 1q25 [5]. After approximately ten years, these studies intersected a totally different discovery path. Linkage studies on families with increased hereditary risk of prostate cancer, identified in 2002 the prostate carcinoma susceptibility gene (Hereditary Prostate Carcinoma 1, HPC-1) on chromosome 1q25, the same of RNASEL location [6]. Different alleles on this locus were associated with higher risk of PCa, such as the R462Q variant, which appeared to provide a 50% risk increase, while homozygosity doubled the risk [7]. This association between a locus behaving as a Tumor Suppressor Gene (TSG) and an Anti-Viral Response (AVR) gene is strongly suggestive of viral involvement in PCa. In the July 2010 presentation at the International Meeting on Muscle Fatigue -which was very critical of the XMRV identification- the sound evidence for viral involvement was emphasized. A logical-inference analysis showed that -most likely- a wrong viral candidate was chosen [1], Fig.1. Subsequent work has vindicated our first prediction (XMRV falsification), but additional work is required to strength the association with another candidate Virus that we propose: MFV (see later) [1]. Several studies have confirmed the RNASEL-HPC1 association [7] [8] [9] [4], but not all [10] [4] of them.

2.2. XMRV discovery

For another five years at the turn of the century, these discoveries on HPC-1 remained just suggestive of a viral involvement in PCA, for a locus -RNASEL- which behaves as a Tumor Suppressor Gene (TSG) -as already indicated by an interesting Editorial by Lengyel, in 1993 [11] and as suggested by others [12] [13]. Then Silverman with colleagues DeRisi and Ganem utilized a micro-array approach (viro-chip) [14], in order to try identifying the responsible virus [3, 15]. The first papers on XMRV appeared at the end of 2006/ beginning of 2007: they showed that XMRV was present at high frequency in patients homozygous for the R462Q allele (i.e., 8/20 or 40%) and that it is a xenotropic retrovirus with similarities with murine leukaemia viruses (MuLV) [16] [15]. Xenotropic retroviruses are endogenous viruses, which cannot infect cells of the original species, while ecotropic viruses do. Typically, endogenous murine retroviruses have been divided into two large families: ecotropic and non-ecotropic retroviruses [17] [18]. Ecotropic retroviruses -being still capable of active infection in the same species, i.e. mouse, cells- are present in only one or just a few copies (0-6) per genome. Their genetics is rather well clarified by several years of research [19]. The structure/genetics of the non-ecotropic retroviruses is more complex, also in view of the fact that they are present in a considerable (40-60) number of copies/genome. In recent years, particularly thanks to the work of J. Coffin and J. Stoye [20], non-ecotropic retroviruses have been clarified and subdivided into three subfamilies: xenotropic (XMP), not capable of replicating in side cells of the same species, polytropic (PMV), which are capable of replicating inside cells of several species including the original (mouse) and modified-polytropic (MPMV), which display altered properties in terms structure/function of the env gene [21] [17] [18]. The experiments, which distinguish among different subfamilies of non-ecotropic mouse retrovi-ruses are: 1. infectivity/replication assays; 2. characterization of their structure by restriction enzyme and/or Southern blotting analysis; 3. complete sequencing [20, 21]. For a more detailed overview of this fascinating but rather complex scientific area, the reader is referred to two excellent review articles by J. Coffin and J. Stoye [17] [18].

2.3. Positive evidence

XMRV was also found integrated inside mesenchimal/stromal cells -rather than in tumour cell genomes- in proximity of genes of cell cycle or hormonal control, which could provide a reasonable link to carcinogenesis [16] [4]. Indeed, such mechanisms variably defined as "promoter insertion" or "insertional mutagenesis" appear to be the most likely involved in chronically (or non-acutely) transforming Retroviruses [22] [23]. This initial report by the discoverer group was followed up a few months later by another PNAS paper, by Schlaberg et al., in which XMRV was associated to approximately 23% of cases by immuno-histochem-istry (IHC), while detection of viral DNA by PCR was quite lower (6%) [24]. Beside this rather surprising finding (since the opposite would be typically expected), this report also slightly contradicted the previous ones, since 1. XMRV was directly identified in the carcinoma cells and not in surrounding mesenchimal/stromal cells, 2. there was no evidence of an association between XMRV positive cases in PCa and RNAse-L involvement by mutation/ lower function, as previously described in the Urisman et al. paper [15, 24]. In that report, 40% of cases which were homozygous for the R462Q variant in RNAse-L were XMRV+ [15]. In the following months of 2010, another group from Emory University in Atalanta (GA) also reported an association between XMRV and PCa, by employing three different and complementary technologies [25]: a) a very sensitive "nested" PCR assay, b) chromosomal fluorescence hybridisation (FISH) and c) very sensitive technology for detection of neutralizing antibodies (the same group and others had previously developed this technique for detecting anti-HIV antibodies) [26] [27] [25]. Also in this report, the serologic assay was the most sensitive, detecting XMRV antibodies in 27.5 % of cases (11/40), while positivity increased in carriers of the R462Q allele (8/20 -also in this study- or 40% of cases, which were RNASEL R462Q homozygous) [25]. Finally, this report confirmed, as in the original paper by Urisman et al., the presence of XMRV in stromal/mesenchimal and not in carcinoma cells [25]. In the same year, another group from Baylor College in Houston (TX) also detected an association between XMRV and PCa in 22% of cases [28]. However, virus was strangely detected in both tumour and normal cells of affected patients and there was no correlation -as in Schlaberg et. al - with RNaseL status [28].

2.4. Negative findings

Together with the appearance of such positive reports, however, a series of studies presenting negative findings started to appear in the literature. Many of these negative reports came from European laboratories, although an initial negative study -often ignored- was from Johns Hopkins University (JHU) in the US [29]: see below. While the issue of XMRV detection in PCa was getting more controversial, another "XMRV-front" opened with the publication in October 2009 of a paper in Science, where Lombardi et al. reported detection of XMRV in 67% (68/101) of Chronic Fatigue Syndrome (CFS) cases [30]. While controls showed much lower detection rates, i.e. 3.7% (8/218), such value (as well as previous ones) was alarming, since it suggested that a few million people may be infected in the general "healthy" population in the US and probably elsewhere [31]. The initial Lombardi et al. paper was followed by larger numbers of negative reports, appearing in the months immediately after its publication: they will not be reviewed extensively in this chapter and the reader is referred instead to the NMD paper [1], with only one exception. In September 2010, Lo et al. published a PNAS paper describing rather frequent association between CFS and a retrovirus different from XMRV: indeed this virus appeared to be polytropic (P-MLV) instead of xenotropic (X-MLV) [32]. While some scientists applauded this novel discovery [33], the PNAS paper was accompanied by an editorial by Andrew Mason's group, in which perplexities about these very findings were expressed [34]. Indeed, despite the relationship between the two viruses, it was extremely difficult to reconcile these findings or even to explain the discovery of XMRV as due to presence of P-MLV instead. In fact, the two viruses are clearly distinguishable by sequencing. Therefore, the idea presented at that time [33]: that the real culprit in CFS would be P-MLV and that the previous detection of XMRV should de facto be considered P-MLV detection, or that either virus could cause the same disease, was simply wrong.

The very first negative report for XMRV in PCa was from Hamburg, DE and was authored (1st) by one of the first co-authors of the original paper by Urisman: Nicole Fischer [35]. This suggests that very similar detection methods were employed in Germany: XMRV was detected only in one non-familiar PCa (of 87) and one control (of 70) sample. Neither one of these cases was homozygous for the R462Q allele [35]. An even more striking negative result was obtained by Hohn and collaborators in Berlin [36], who did not detect a single positive case among 589 PCa patients tested: this study employed a sensitive nested PCR detection, RT-PCR for gag sequences as well as serology for XMRV-specific antibodies [36]. A number of patients (76) were studied for the RNASEL allele and 12.9% scored positive [36]. Similar negative results were published in additional studies from Ireland (139 cases) [37], Holland (74 sporadic cases) [38], Mexico (55 cases) [39], USA (over 800 patients from a collaborative effort between Baylor, Johns Hopkins etc.) [40] and UK (437 patients from UK, Korea and Thailand) [41]. In the last study, a few patients scored positive: for example 2 out of 6 of Thailand's patients were positive, potentially reaching a score of 33%. However, evidence of contamination started emerging in this British International study: some of the amplified DNA did not contain a 24 bp deletion which is a hallmark of XMRV and other evidence suggested instead presence of P-MLV (as in the previous paper by Lo et al. on CFS) [41] [32]. A few assays, specific for contamination by mouse DNA, were therefore run to confirm identity of specimens. A very sensitive assay for Intracisternal A-type particles (IAPs) and mouse mitocondrial DNA was completely concordant with XMRV presence, clearly indicating presence of contamination [41]. Therefore, this 2010 paper by Robinson should have already signalled a red-flag warning for XMRV research [41].

2.5. Strength of RNASEL - HPC-1 paradigm

At the International Congress on Muscle Fatigue in 2010, I strongly criticized the association between PCa and XMRV, on the basis of such negative findings, most of which had been already published in the literature (July 2010). My analysis at the congress extended to the technology employed, thus suggesting that the viro-chip assay was -most likely- the source of error [1]. Still, data on the RNase-L association with HPC-1 were indicative of viral involvement. Contrary to the situation in PCa, in which a few independent reports confirmed XMRV presence, while they were contradicted by a limited number of studies, CFS association with this virus was essentially based upon the unique paper by Lombardi et al. in 2009, somehow overwhelmed by a plethora of negative reports [1]. However, also in CFS, the case for the likely presence of an infectious agent, most probably a virus, can be made. This is particularly clear, in view of the presence of "micro-epidemics", often associated with CFS onset [1]. The rather strong evidence for a previous virus infection accompanied by the dramatic personal histories of CFS onset in thousands of patients could explain, but certainly NOT justify, the attachment of some patient-groups to the XMRV hypothesis, sometimes referred in the media as mass-hysteria [224]. We will later discuss whether the viral hypothesis should be completely dismissed in view of XMRV falsification or whether additional viral candidates should be investigated (see section 3).

2.6. XMRV controversy: looking back through 3 major Editorials

After 2010, the majority of XMRV reports documented negative results either in PCa or in CFS cases. Yet, the heated debate could have continued much longer, with some extreme defence of the XMRV hypothesis (J. Mikovitz) and with a more balanced overview of the criticisms by R. Silverman (see for example, his excellent review in Nature Reviews of Urology, extensively discussing criticisms) [4]. Examples of debates on possible infectious agents present in human cancers are abundant in the literature: for PCa, HPVs are still extensively discussed as potential etiological agents or onset-cofactors see discussion in Sections 4.3 (3) and 4.3.1 (c). What or who was capable of rescinding the "Gordian Knot" of XMRV cancer/CFS association ? If we want to name a single scientist this is certainly John Coffin, although he extensively collaborated with other groups, especially with the group of S. Pa-thak. And yet, Coffin himself had written with J. Stoye in Science, accompanying one of the first papers on XMRV discovery -that of Lombardi et al. on the CFS association [30]- a positive editorial comment, which emphasized the future potential of such discovery [31].

i. It may be instructive in this respect to re-analyse -so to speak: after the facts- the three major editorials, which accompanied the three major discovery-articles associated with XMRV. The first is the article by Dong et al. in PNAS at the beginning of 2007 [16], therefore immediately after publication of the Urisman et al. paper (December 2006). This article really gave credibility to the XMRV hypothesis, by showing that the virus was: 1. capable of replication in human cells, once a com plete copy of the provirus was cloned and reconstructed; 2. responsive to the IFN pathway, as it had been predicted in view of the RNase L mutations; 3. uses a specific receptor, XPR-1 (therefore capable of mediating entrance for both xenotropic and polytropic retroviruses) for infecting human cells; 4. in three cases analysed, XMRV was integrated in tumour cells in regions surrounding potentially interesting/important genes, in two cases next to transcription factor genes (CREB and NFAT) and in the third, next to a hormone response gene, causing inhibition of androgen receptor trans-activation (APPB2/PAT1/ARA67). The accompanying editorial, by retro-virologist Hung Fan, is certainly the most cautious and critical of the three editorials [43]. Although underlying the potential importance of these findings, Fan clearly indicated that they were generating more questions than answers and that only by answering such questions could the XMRV hypothesis be strengthened or proven [43]. In one sentence, his cautionary criticism was particularly evident: "However, another possibility is that XMRV is not causal to PC, but reflective of the reduced antiviral status of RNase L QQ individuals; another novel virus whose sequences were not detectd by the ViroChip might be the relevant agent" (bold characters are my additions) [43].

ii. The second fundamental paper for the XMRV hypothesis was the one by Lombardi et al. (2009), in which an astonishing 67% XMRV presence was documented in Chronic Fatigue Syndrome samples [30]. The paper was already briefly described, as well as the strong critical reaction it has generated, although this section is covered in more depth in the NMD review (see [1]) [30]. Surprisingly, the accompanying editorial written by John Coffin and Jonathan Stoye, appears to emphasize the positive aspects of these findings, rather than caution the readers about potential pitfalls, such as contaminations/artefacts [31]. It is apparent that the two Editorialists, among the major experts in mouse retro-virology, believed in 2009 that XMRV had strong connection to CFS, although it should be reminded that other viral infections have been previously associated with CFS (EBV, HHSV-6, HTLV etc., see [1]) [31]. And yet Coffin's with Pathak's groups eventually "put the nails into the XMRV coffin one by one" [44]. Far from being a "changing party" episode, reassessment of scientific data and even of personal believes is an essential and intrinsic process of scientific endeavour. One of the greatest epistemologists of past century, Karl Popper, has identified in the process of empirical falsification one of the essential logical characters of science in western world. In his "All Life is Problem Solving" Popper suggests that our scientific theories develop as an evolutionary (almost Darwinian) process, in which it is however falsification rather than verification the discriminating instrument (Occam's razor). Therefore, it is just natural and physiological that today in science, hypotheses and theories are continuously re-evaluated and reassessed, although in this process strong intellectual honesty and courage are also needed. Most likely, in 2009 Coffin/Stoye positively reacted and were convinced by 1. the fact that XMRV demonstrated a clear homology to MLV endogenous sequences, but different enough and with constant/homologous difference (approximately 10% throughout the viral genome) to let us believe that this was a totally new isolate. 2. The fact that all XMRV isolates detected showed strong ho-mology among each other (less than 30 nucleotide variations in a genome of over 8000 bp.s), could be again evidence of an exogenous infecting agent (but also a contaminating virus). 3. Somehow, the general homology of XMRV with endogenous MLVs of approx. 90% may have been misleading still in 2009, since it might have suggested a mechanism of constant mutation accrual, as in phylogenetic analysis, of which the two editorialists are great experts [31]. In XMRV, however, recombination plays a major and determining role, as it was initially suggested in a PNAS editorial one year later, by Andrew Mason and colleagues (accompanying the third XMRV/MLV paper by Lo et al.) [34] [32].

iii. Lo's paper initially appeared (or it was presented as) confirmatory of the infection hypothesis in CFS, since a murine retroviral sequence was detected in 86.5% of cases and only 7% of controls [32] [34]. The viral sequences however were not identical or very similar to XMRV, as previously reported, and appeared to be related to endogenous Polytropic retroviruses (PMLV). This generated some scepticism, as in previous work the viral sequences had little difference from the prototype retrovi-rus -XMRV. In his editorial, Mason underlines some discrepancies and yet does not clearly indicate that the finding of one xenotropic and one polytropic retroviruses are incompatible [34]. In other words, a general misconception could be -and apparently was- generated: there is an endogenous-like mouse retrovirus infecting cells in prostate carcinoma and CFS. In this scenario, apparently it didn't really matter whether it was marked with a P or with a X (for Polytropic and Xenotropic): the relevant and important point was that some type of murine endogenous-like retro-virus was infecting Homo sapiens in such disorders [34]. The paper by Ila Singh was also in line with such (mis-)interpretation [33]. On the other hand, as also pointed out in the previous editorial by Coffin and Stoye, the strength of the original XMRV hypothesis laid in the fact that all the isolates were similar to each other, although the prototype of XMRV appeared to be unique, different from any retrovirus known at that time [31]. Furthermore, Mason group's editorial suggested that, while the issue of which retrovirus exactly is present in PCa and/or CFS was being solved, a realistic and effective strategy could have been to test already potential therapeutic approaches with antiretroviral agents [34]. Again, such attitude is logically biased by the caveat that there was no firm evidence at that time for the real involvement of a retrovirus in both human conditions: this has been completely confirmed now by XMRV falsification. In fact, the paper by Lo et al. was rather good evidence against involvement of a retrovirus in both human conditions, since it suggested that contamination could be the cause [32]. Contamination, although denied in Lo's paper by a series of counter evidences, could explain the association with an endogenous murine polytropic retrovirus and, by extension, also with XMRV [32]. Andrew Mason group's editorial also emphasized the fact XMRV sequences appeared to be the result of recombinatory events [34]. They observed that in XMRV, while the 5' portion of its genome shares great homology to polytropic murine retroviruses, the 3' end is most similar to endogenous xenotropic MLV [34].

2.7. XMRV falsification

This observation, that inescapably leads to presence of recombination, was further developed approximately one year later in a seminal article by the groups of J. Coffin and S. Pa-thak [45]. In this Science paper in May 2011, Paprotka et al. convincingly showed that XMRV was generated by recombination during passage of the original tumor cells in nude mice [45]. The creation of human cell line 22 Rv1 was reported in 1999 after several passages by xenotransplantation, starting from 1993. The late passages /established cell line display presence of several copies of integrated XMRV provirus as well as high titers of virus production (1010-1011 PFU/ml). However, Paprotka et al. established a few essential and undermining criticisms: 1. First of all, fully infectious XMRV could not be detected in the original tumor explant (less than 1 copy/200 cells). 2. Second, two regions of strong homology with endogenous viruses could be detected: the 5'-end (called preXMRV-2) displays strong homology to PMLV endogenous sequences, while the 3'-end region (called PreXMRV-1) is most similar to an endogenous xenotropic retrovirus (XMLV). 3. Third, highly infectious "recombinant" XMRV started to appear in xenografts passaged in nude mice since 1996, i.e., three years after initial establishment of this tumour xenografts. This strongly suggests that infectious XMRV was created or has infected these cells between 1993 and 1996. 4. Fourth, the original nude mice strains utilized in xenotransplantation experiments did contain as endogenous viruses both the endogenous xenotropic virus (pre-XMRV-1, present in 6 out of 48 tested and typical of European mouse strains) as well as the endogenous PMLV (preXMRV-2, present in 25 out of 48 tested and typical of Asian mouse strains). 5. Fifth, the overall structure of the infectious XMRV could be explained by six recombinatory events between the two viruses: preXMRV-2 and preXMRV-1. Indeed, recombination is known to frequently occur during retrovirus replication, due to a polymerase (i.e., reverse transcriptase) switching between two different templates, therefore a mechanism of "copy-choice" as compared to the classical mechanism of "cut-and-paste" typical of general recombination [45] [46]. 6. Finally, the presence of a unique XMRV structure after so many recombinatory events strongly indicates that this "creation" occurred only once, most likely during xenograft passaging into nude mice [45]. The paper by Poprotka et al. therefore concluded the "XMRV Odyssey" with a most logical and well proven explanation and XMRV-falsification [45].

Additional evidence against XMRV as an exogenous virus infecting the human species were also obtained by the group of Jay Levy, who analysed some of the same CFS samples initially studies by Lombardi et al. Since these patients, initially reported as XMRV-positive, were found devoid of this retrovirus, this finding once more strengthened the evidence for contamination in positive samples [47]. A series of subsequent papers then reported evidence for contamination [45] [44] [48] [49] [50] in: 1. PCR reagents (even Taq polymerase) employed for XMRV detection; 2. microtomes or blades for tumours sections (even one year after the initial experiment); 3. contamination of several cell lines, beside the original 22Rv1. Prostate carcinoma cells lack the APOBEK-GA3 activity and are therefore susceptible to XMRV infection, while other human cells -for example human lymphocytes- appear to be highly resistant in view of the strong mutagenic activity of APOBEK-GA3.

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