We recently reported identification of a previously undescribed gammaretrovirus genome, xenotropic murine leukemia virusrelated virus (XMRV), in prostate cancer tissue from patients homozygous for a reduced activity variant of the antiviral enzyme RNase L. Here we constructed a full-length XMRV genome from prostate tissue RNA and showed that the molecular viral clone is replication-competent. XMRV replication in the prostate cancer cell line DU145 was sensitive to inhibition by IFN-␤. However, LNCaP prostate cancer cells, which are deficient in JAK1 and RNase L, were resistant to the effects of IFN-␤ against XMRV. Furthermore, DU145 cells rendered deficient in RNase L with siRNA were partially resistant to IFN inhibition of XMRV. Expression in hamster cells of the xenotropic and polytropic retrovirus receptor 1 allowed these cells to be infected by XMRV. XMRV provirus integration sites were mapped in DNA isolated from human prostate tumor tissue to genes for two transcription factors (NFATc3 and CREB5) and to a gene encoding a suppressor of androgen receptor transactivation (APPBP2/PAT1/ARA67). Our studies demonstrate that XMRV is a virus that has infected humans and is susceptible to inhibition by IFN and its downstream effector, RNase L.cancer ͉ RNase L ͉ xenotropic murine leukemia virus-related virus A diverse range of mammalian species are susceptible to infections by viruses from the gammaretrovirus genus of Retroviridae (1). Examples of these simple viruses whose genomes include gag, pro, pol, and env genes only are murine leukemia virus (MLV), feline leukemia virus, koala retrovirus, and gibbon ape leukemia virus. These viruses are responsible for leukemogenesis and other diseases in their respective host species (1-3). However, until recently evidence of authentic infections of humans by gammaretroviruses was lacking. We reported in 2006 identification of viral genomes for a previously undescribed gammaretrovirus, termed xenotropic MLV-related virus (XMRV), in a subset of men with prostate cancer (4). The discovery of XMRV followed investigations of the role of the antiviral enzyme RNase L in hereditary prostate cancer, a disease in which tumors arise in three or more first-degree relatives (5). The human RNase L gene (RNASEL) was initially proposed as a candidate for the hereditary prostate cancer 1 (HPC1) gene based on a positional cloning/candidate gene method (6).RNase L is a regulated endoribonuclease for single-stranded RNA that functions in the IFN antiviral response (7,8). IFN treatment of cells induces a family of 2Ј-5Ј oligoadenylate synthetases that produce 5Ј-phosphorylated, 2Ј-5Ј-linked oligoadenylates (2-5A) from ATP in response to stimulation by viral dsRNA. 2-5A activates the preexisting, latent, and ubiquitous RNase L, resulting in degradation of viral and cellular RNA. Sustained activation of RNase L leads to apoptosis, a function consistent with a role in the suppression of tumor growth (9). Although mice lacking RNase L do not spontaneously develop tumors at higher rates than wild-typ...

SUMMARY In mice, clonal tracking of hematopoietic stem cells has revealed variations in repopulation characteristics. However, it is unclear whether similar properties apply in primates. Here, we examined this issue through tracking of thousands of hematopoietic stem and progenitor cells (HSPCs) in rhesus macaques for up to 12 years. Approximately half of the clones analyzed contributed to long-term repopulation (over 3–10 years) and likely represent self-renewing hematopoietic stem cells (HSCs), while the remainder contributed primarily for the first year. The long-lived clones could be further subdivided into functional groups contributing primarily to myeloid, lymphoid or both lineages. Over time, the 4–10% of clones with robust dual lineage contribution predominated in repopulation capacity. HSPCs expressing a CCR5 shRNA transgene behaved similarly to controls. Our study therefore documents HSPC behavior in a clinically-relevant model over a long time frame, and provides a substantial system-level dataset that is a reference point for future work.

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Xenotropic murine leukemia virus-related virus (XMRV) is a new human gammaretrovirus identified inprostate cancer tissue from patients homozygous for a reduced-activity variant of the antiviral enzyme RNase L. Neither a casual relationship between XMRV infection and prostate cancer nor a mechanism of tumorigenesis has been established. To determine the integration site preferences of XMRV and the potential risk of proviral insertional mutagenesis, we carried out a genome-wide analysis of viral integration sites in the prostate cell line DU145 after an acute XMRV infection and compared the integration site pattern of XMRV with those found for murine leukemia virus and two human retroviruses, human immunodeficiency virus type 1 and human T-cell leukemia virus type 1. Among all retroviruses analyzed, XMRV has the strongest preference for transcription start sites, CpG islands, DNase-hypersensitive sites, and gene-dense regions; all are features frequently associated with structurally open transcription regulatory regions of a chromosome. Analyses of XMRV integration sites in tissues from prostate cancer patients found a similar preference for the aforementioned chromosomal features. Additionally, XMRV integration sites in cancer tissues were associated with cancer breakpoints, common fragile sites, microRNA, and cancer-related genes, suggesting a selection process that favors certain chromosomal integration sites. In both acutely infected cells and cancer tissues, no common integration site was detected within or near proto-oncogenes or tumor suppressor genes. These results are consistent with a model in which XMRV may contribute to tumorigenicity via a paracrine mechanism.Prostate cancer is the most common noncutaneous cancer diagnosed in men in developed countries and is responsible for the deaths of approximately 30,000 men per year in the United States (43). Despite its impact on male health, the molecular mechanisms involved in the pathogenesis of prostate cancer, particularly the events contributing to initiation and progression, remain relatively unknown in comparison with those for other common cancers. Epidemiological studies of kindreds with hereditary prostate cancer, who often display early-onset disease and account for 9% of all cases (16), identified HPC1 as a susceptibility locus for prostate cancer (94). HPC1 is linked to RNASEL, which encodes a regulated endoribonuclease for single-stranded RNA and functions in the antiviral action of interferon (IFN) (15, 17). In response to stimulation by viral double-stranded RNA, IFN treatment of cells induces a family of 2Ј-5Ј oligoadenylate synthetases that produce 2Ј-5Ј-linked oligoadenylates, which then activate the latent and ubiquitous protein RNase L, resulting in degradation of viral and cellular RNA and apoptosis induction (112). Several germ line variants of HPC1 and RNASEL have been observed in hereditary prostate cancer (91), including a common (35% allelic frequency) missense variant of RNase L in which a G-to-A transition at nucleotide position 13...

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Until very recently, quiescent CD4؉ T cells were thought to be resistant to human immunodeficiency virus (HIV) infection. Subsequent studies, attempting to fully elucidate the mechanisms of resistance, showed that quiescent cells could become infected by HIV at low efficiency and form a latently infected population. In this study, we set out to identify the sites of viral integration and to assess the efficiency of the overall integration process in quiescent cells. Based on our results, HIV integration in quiescent CD4؉ T cells occurs in sites similar to those of their prestimulated counterparts. While site selections are similar, the integration process in quiescent cells is plagued by the formation of high levels of incorrectly processed viral ends and abortive two-long-terminal-repeat circles. Quiescent CD4ϩ T cells have been shown to be resistant to human immunodeficiency virus (HIV) infection, and the resistance is characterized by incomplete reverse transcription (82,83). However, the permissiveness of other nondividing cell types, such as resting T cells and macrophages, raised further questions regarding the nature of the block (10,26,56,65,67,(69)(70)(71). Later studies further elucidated which subsets of resting cells were refractory to infection. Truly quiescent cells in the G 0/1a phase were resistant to infection, while cells in the G 1b phase, characterized by high levels of RNA synthesis but not DNA synthesis, were susceptible to infection (43). These combined studies suggested that certain nondividing cells could support a productive infection. Subsequent studies were aimed at further examining the different steps of the viral life cycle in quiescent cells, as well as identifying potential cellular factors or the lack thereof that may block infection. One potential block to infection was hypothesized to be the lack of raw materials such as nucleotides. Treatment of quiescent cells with nucleosides resulted in increased reverse transcription but did not lead to a productive infection following stimulation of the cells (42, 61), suggesting that other factors may contribute to the resistance to productive infection. Recent studies examined the presence or absence of cellular factors responsible for the block in quiescent CD4 ϩ T cells. Both Murr1 and APOBEC3G have been shown to influence the viral life cycle in quiescent CD4 ϩ T cells (18, 29). However neither factor fully explains the lack of rescue of productive infection if quiescent cells are stimulated subsequent to infection.A more detailed examination of the viral life cycle in quiescent CD4 ϩ T cells can shed more light on the nature of the block presented by quiescent CD4 ϩ T cells. A series of recent studies looking at the different stages of the HIV life cycle in quiescent cells have further supported data from our earlier work (82, 83). More specifically, it has been shown that reverse transcription is inefficient in quiescent cells, generating fulllength viral transcripts that are very labile (half-life of 1 day) but are integration co...

BackgroundHow a potentially diverse population of hematopoietic stem cells (HSCs) differentiates and proliferates to supply more than 1011 mature blood cells every day in humans remains a key biological question. We investigated this process by quantitatively analyzing the clonal structure of peripheral blood that is generated by a population of transplanted lentivirus-marked HSCs in myeloablated rhesus macaques. Each transplanted HSC generates a clonal lineage of cells in the peripheral blood that is then detected and quantified through deep sequencing of the viral vector integration sites (VIS) common within each lineage. This approach allowed us to observe, over a period of 4-12 years, hundreds of distinct clonal lineages.ResultsWhile the distinct clone sizes varied by three orders of magnitude, we found that collectively, they form a steady-state clone size-distribution with a distinctive shape. Steady-state solutions of our model show that the predicted clone size-distribution is sensitive to only two combinations of parameters. By fitting the measured clone size-distributions to our mechanistic model, we estimate both the effective HSC differentiation rate and the number of active HSCs.ConclusionsOur concise mathematical model shows how slow HSC differentiation followed by fast progenitor growth can be responsible for the observed broad clone size-distribution. Although all cells are assumed to be statistically identical, analogous to a neutral theory for the different clone lineages, our mathematical approach captures the intrinsic variability in the times to HSC differentiation after transplantation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0191-8) contains supplementary material, which is available to authorized users.

Autocatalytic proteolytic cleavage is a frequently observed post-translational modification in proteins. Cephalosporin acylase (CA) is a recently identified member of the N-terminal hydrolase family that is activated from an inactive precursor by autoproteolytic processing, generating a new N-terminal residue, which is either a Ser or a Thr. The N-terminal Ser or Thr becomes a nucleophilic catalytic center for intramolecular and intermolecular amide cleavages. The gene structure of the open reading frame of CAs generally consists of a signal peptide followed by the ␣-subunit, a spacer sequence, and the ␤-subunit, which are all translated into a single polypeptide chain, the CA precursor. The precursor is post-translationally modified into an active heterodimeric enzyme with ␣-and ␤-subunits, first by intramolecular cleavage and second by intermolecular cleavage. We solved the first CA precursor structure (code 1KEH) from a class I CA from Pseudomonas diminuta at a 2.5-Å resolution that provides insight into the mechanism of intramolecular cleavage. A conserved water molecule, stabilized by four hydrogen bonds in unusual pseudotetrahedral geometry, plays a key role to assist the OG atom of Ser 1␤ to generate a strong nucleophile. In addition, the site of the secondary intermolecular cleavage of CA is proposed to be the carbonyl carbon of Gly 158␣ (Kim, S., and Kim, Y., (2001) J. Biol. Chem., 276, 48376 -48381), which is different from the situation in two other class I CAs.