Self-Repairing Herpesvirus Saimiri Deletion Variants.

Herpesvirus saimiri (HVS) is discussed as a possible vector in gene therapy. In order to create a self-repairing HVS vector, the F plasmid vector moiety of the bacterial artificial chromosome (BAC) was transposed via Red recombination into the virus genes ORF22 or ORF29b, both important for virus replication. Repetitive sequences were additionally inserted, allowing the removal of the F-derived sequences from the viral DNA genome upon reconstitution in permissive epithelial cells. Moreover, these self-repair-enabled BACs were used to generate deletion variants of the transforming strain C488 in order to minimalize the virus genome. Using the en passant mutagenesis with two subsequent homologous recombination steps, the BAC was seamlessly manipulated. To ensure the replication capacity in permissive monkey cells, replication kinetics for all generated virus variants were documented. HVS variants with increased insert capacity reached the self-repair within two to three passages in permissive epithelial cells. The seamless deletion of ORFs 3/21, 12-14, 16 or 71 did not abolish replication competence. Apoptosis induction did not seem to be altered in human T cells transformed with deletion variants lacking ORF16 or ORF71. These virus variants form an important step towards creating a potential minimal virus vector for gene therapy, for example, in human T cells.

Allosteric regulation of noncoding RNA function by microRNAs.

HSUR1 and HSUR2, two noncoding RNAs expressed by the oncogenic Herpesvirus saimiri, bind host microRNAs miR-142-3p, miR-16, and miR-27 with different purposes. While binding of miR-27 to HSUR1 triggers the degradation of the microRNA, miR-16 is tethered by HSUR2 to target host mRNAs to repress their expression. Here we show that the interaction with miR-142-3p is required for the activity of both HSURs. Coimmunoprecipitation experiments revealed that miR-142-3p allosterically regulates the binding of miR-27 and miR-16 to HSUR1 and HSUR2, respectively. The binding of two different miRNAs to each HSUR is not cooperative. HSURs can be engineered to be regulated by other miRNAs, indicating that the identity of the binding miRNA is not important for HSUR regulation. Our results uncover a mechanism for allosteric regulation of noncoding RNA function and a previously unappreciated way in which microRNAs can regulate gene expression.

Learning noncoding RNA biology from viruses.

Insights into interactions between viral factors and the cellular machinery usually lead to discoveries concerning host cell biology. Thus, the gene expression field has historically relied on viral model systems to discover mechanisms underlying different cellular processes. In recent years, the functional characterization of the small nuclear noncoding RNAs expressed by the oncogenic Herpesvirus saimiri, called HSURs, resulted in the discovery of two mechanisms for the regulation of gene expression. HSUR1 and HSUR2 associate with host microRNAs, which are small noncoding RNAs that broadly regulate gene expression by binding to messenger RNAs. HSUR1 provided the first example of a process known as target-directed miRNA degradation that operates in cells to regulate miRNA populations. HSUR2 functions as a miRNA adaptor, uncovering an entirely new, indirect mechanism by which miRNAs can inhibit mRNA function. Here, I review the path that led to these discoveries and their implications and postulate new exciting questions about the functions of these fascinating viral noncoding RNAs.

Viral miRNA adaptor differentially recruits miRNAs to target mRNAs through alternative base-pairing.

HSUR2 is a viral non-coding RNA (ncRNA) that functions as a microRNA (miRNA) adaptor. HSUR2 inhibits apoptosis in infected cells by recruiting host miRNAs miR-142-3p and miR-16 to mRNAs encoding apoptotic factors. HSUR2's target recognition mechanism is not understood. It is also unknown why HSUR2 utilizes miR-16 to downregulate only a subset of transcripts. We developed a general method for individual-nucleotide resolution RNA-RNA interaction identification by crosslinking and capture (iRICC) to identify sequences mediating interactions between HSUR2 and target mRNAs in vivo. Mutational analyses confirmed identified HSUR2-mRNA interactions and validated iRICC as a method that confidently determines sequences mediating RNA-RNA interactions in vivo. We show that HSUR2 does not display a 'seed' region to base-pair with most target mRNAs, but instead uses different regions to interact with different transcripts. We further demonstrate that this versatile mode of interaction via variable base-pairing provides HSUR2 with a mechanism for differential miRNA recruitment.

Evaluation of Cyclin D1 as a Discriminatory Immunohistochemical Biomarker for Idiopathic Pulmonary Fibrosis.

BACKGROUND: Distinction of idiopathic pulmonary fibrosis (IPF) from other chronic fibrosing interstitial pneumonitides, such as hypersensitivity pneumonitis (HP) and connective tissue diseases, is critical due to varied biological and clinical outcomes. However, their histologic overlaps often pose diagnostic challenges. A recent study suggested an association of herpesvirus saimiri infection with IPF. Productive viral infection is associated with coexpression of pirated mammalian protein cyclin D1, shown to be overexpressed by immunohistochemistry (IHC) in the regenerating alveolar epithelium in IPF but not in normal lungs. We evaluated the diagnostic utility of cyclin D1 to discriminate between IPF and other fibrosing interstitial lung diseases. MATERIALS AND METHODS: A retrospective study of cyclin D1 IHC expression in 27 consecutive cases of chronic fibrosing interstitial lung diseases from 2011 to 2017: 12 usual interstitial pneumonia (UIP) pattern; 5 nonspecific interstitial pneumonia pattern; 3 HP pattern; 7 unclassifiable was performed. Five cases of normal lung obtained from lobectomy specimen for malignancy are included as control. Immunoreactivity was graded semiquantitatively on a scale of 0 to 3. RESULTS: Cyclin D1 staining was uniformly strongly positive in all cases evaluated in the study, particularly in proliferating type II pneumocytes in the region of fibrosing areas. There was no statistical difference in the extent of cyclin D1 expression between UIP and non-UIP groups (2.7 vs. 2.5) and IPF versus non-IPF groups (2.7 vs. 2.4). Cyclin D1 expression is lower in control group compared with UIP groups (1.2 vs. 2.7). CONCLUSIONS: Cyclin D1 is not a specific marker of UIP pattern/IPF. The high expression of cyclin D1 in lung tissue of fibrosing interstitial pneumonitides regardless of etiology most likely correlates with proliferation in type II pneumocytes.

Structural identification of conserved RNA binding sites in herpesvirus ORF57 homologs: implications for PAN RNA recognition.

Kaposi's sarcoma-associated herpesvirus (KSHV) transcribes a long noncoding polyadenylated nuclear (PAN) RNA, which promotes the latent to lytic transition by repressing host genes involved in antiviral responses as well as viral proteins that support the latent state. KSHV also expresses several early proteins including ORF57 (Mta), a member of the conserved multifunctional ICP27 protein family, which is essential for productive replication. ORF57/Mta interacts with PAN RNA via a region termed the Mta responsive element (MRE), stabilizing the transcript and supporting nuclear accumulation. Here, using a close homolog of KSHV ORF57 from herpesvirus saimiri (HVS), we determined the crystal structure of the globular domain in complex with a PAN RNA MRE, revealing a uracil specific binding site that is also conserved in KSHV. Using solution NMR, RNA binding was also mapped within the disordered N-terminal domain of KSHV ORF57, and showed specificity for an RNA fragment containing a GAAGRG motif previously known to bind a homologous region in HVS ORF57. Together these data located novel differential RNA recognition sites within neighboring domains of herpesvirus ORF57 homologs, and revealed high-resolution details of their interactions with PAN RNA, thus providing insight into interactions crucial to viral function.

Novel roles for Sm-class RNAs in the regulation of gene expression.

Viruses masterfully regulate host gene expression during infection. Many do so, in part, by expressing non-coding RNAs. Recent work has shown that HSUR 2, a viral non-coding RNA expressed by the oncogenic Herpesvirus saimiri, regulates mRNA expression through a novel mechanism. HSUR 2 base pairs with both target mRNAs and host miRNAs in infected cells. This results in HSUR 2-dependent recruitment of host miRNAs and associated Ago proteins to target mRNAs, and the subsequent destabilization of target mRNAs. Using this mechanism, this virus regulates key cellular pathways during viral infection. Here I discuss the evolution of our thinking about HSUR function and explore the implications of recent findings in relation to the current views on the functions of interactions between miRNAs and other classes of non-coding RNAs, the potential advantages of this mechanism of regulation of gene expression, and the evolutionary origin of HSUR 2.

A viral Sm-class RNA base-pairs with mRNAs and recruits microRNAs to inhibit apoptosis.

Viruses express several classes of non-coding RNAs; the functions and mechanisms by which most of these act are unknown. Herpesvirus saimiri, a γ-herpesvirus that establishes latency in the T cells of New World primates and has the ability to cause aggressive leukaemias and lymphomas in non-natural hosts, expresses seven small nuclear uracil-rich non-coding RNAs (called HSURs) in latently infected cells. These HSURs associate with Sm proteins, and share biogenesis and structural features with cellular Sm-class small nuclear RNAs. One of these HSURs (HSUR2) base-pairs with two host cellular microRNAs (miR-142-3p and miR-16) but does not affect their abundance or activity, which suggests that its interactions with them perform alternative functions. Here we show that HSUR2 also base-pairs with mRNAs in infected cells. We combined in vivo psoralen-mediated RNA-RNA crosslinking and high-throughput sequencing to identify the mRNAs targeted by HSUR2, which include mRNAs that encode retinoblastoma and factors involved in p53 signalling and apoptosis. We show that HSUR2 represses the expression of target mRNAs and that base-pairing between HSUR2 and miR-142-3p and miR-16 is essential for this repression, suggesting that HSUR2 recruits these two cellular microRNAs to its target mRNAs. Furthermore, we show that HSUR2 uses this mechanism to inhibit apoptosis. Our results uncover a role for this viral Sm-class RNA as a microRNA adaptor in the regulation of gene expression that follows precursor mRNA processing.

Constitutive activation of T cells by γ2-herpesviral GPCR through the interaction with cellular CXCR4.

Members of the herpesviral family use multiple strategies to hijack infected host cells and exploit cellular signaling for their pathogenesis and latent infection. Among the most intriguing weapons in the arsenal of pathogenic herpesviruses are the constitutively active virally-encoded G protein-coupled receptors (vGPCRs). Even though vGPCRs contribute to viral pathogenesis such as immune evasion and proliferative disorders, the molecular details of how vGPCRs continuously activate cellular signaling are largely unknown. Here, we report that the vGPCR of Herpesvirus saimiri (HVS), an oncogenic γ2-herpesvirus, constitutively activates T cells via a heteromeric interaction with cellular CXCR4. Constitutive T cell activation also occurs with expression of the vGPCR of Kaposi's sarcoma-associated herpesvirus (KSHV), but not the vGPCR of Epstein-Barr virus. Expression of HVS vGPCR down-regulated the surface expression of CXCR4 but did not induce the degradation of the chemokine receptor, suggesting that vGPCR/CXCR4 signaling continues in cytosolic compartments. The physical association of vGPCR with CXCR4 was demonstrated by proximity ligation assay as well as immunoprecipitation. Interestingly, the constitutive activation of T cells by HVS vGPCR is independent of proximal T cell receptor (TCR) signaling molecules, such as TCRß, Lck, and ZAP70, whereas CXCR4 silencing by shRNA abolished T cell activation by vGPCRs of HVS and KSHV. Furthermore, previously identified inactive vGPCR mutants failed to interact with CXCR4. These findings on the positive cooperativity of vGPCR with cellular CXCR4 in T cell activation extend our current understanding of the molecular mechanisms of vGPCR function and highlight the importance of heteromerization for GPCR activity.

Transduction of Herpesvirus saimiri-Transformed T Cells with Exogenous Genes of Interest.

Human T cells can be transformed and expanded with herpesvirus saimiri (HVS). HVS-transformed T cells from patients have facilitated the study of a broad range of primary immunodeficiencies (PID) in which T-cell development or function is altered. However, the utility of HVS-transformed T cells for genetic studies has been limited by technical challenges in the expression of exogenous genes, including wild-type or mutant alleles. A novel, gamma retrovirus-based method for the simple and reliable transduction, purification, and study of HVS-transformed T cells is described. © 2016 by John Wiley & Sons, Inc.

Host miRNA degradation by Herpesvirus saimiri small nuclear RNA requires an unstructured interacting region.

Herpesvirus saimiri, an oncogenic herpesvirus, during latency produces seven small nuclear RNAs, called the Herpesvirus saimiri U RNAs (HSUR1-7). HSUR1 mediates degradation of the host microRNA, miR-27, via a process that requires imperfect base-pairing. The decreased levels of miR-27 lead to prolonged T-cell activation and likely contribute to oncogenesis. To gain insight into HSUR1-mediated degradation of miR-27, we probed the in vivo secondary structure of HSUR1 and coupled this with bioinformatic structural analyses. The results suggest that HSUR1 adopts a conformation different than previously believed and that the region complementary to miR-27 lacks stable structure. To determine whether HSUR1 structural flexibility is important for its ability to mediate miR-27 degradation, we performed structurally informative mutagenic analyses of HSUR1. HSUR1 mutants in which the miR-27 binding site sequence is preserved, but sequestered in predicted helices, lose their ability to decrease miR-27 levels. These results indicate that the HSUR1 miR27-binding region must be available in a conformationally flexible segment for noncoding RNA function.

Modulating p56Lck in T-Cells by a Chimeric Peptide Comprising Two Functionally Different Motifs of Tip from Herpesvirus saimiri.

The Lck interacting protein Tip of Herpesvirus saimiri is responsible for T-cell transformation both in vitro and in vivo. Here we designed the chimeric peptide hTip-CSKH, comprising the Lck specific interacting motif CSKH of Tip and its hydrophobic transmembrane sequence (hTip), the latter as a vector targeting lipid rafts. We found that hTip-CSKH can induce a fivefold increase in proliferation of human and Aotus sp. T-cells. Costimulation with PMA did not enhance this proliferation rate, suggesting that hTip-CSKH is sufficient and independent of further PKC stimulation. We also found that human Lck phosphorylation was increased earlier after stimulation when T-cells were incubated previously with hTip-CSKH, supporting a strong signalling and proliferative effect of the chimeric peptide. Additionally, Lck downstream signalling was evident with hTip-CSKH but not with control peptides. Importantly, hTip-CSKH could be identified in heavy lipid rafts membrane fractions, a compartment where important T-cell signalling molecules (LAT, Ras, and Lck) are present during T-cell activation. Interestingly, hTip-CSKH was inhibitory to Jurkat cells, in total agreement with the different signalling pathways and activation requirements of this leukemic cell line. These results provide the basis for the development of new compounds capable of modulating therapeutic targets present in lipid rafts.

Convergent capture of retroviral superantigens by mammalian herpesviruses.

Horizontal gene transfer from retroviruses to mammals is well documented and extensive, but is rare between unrelated viruses with distinct genome types. Three herpesviruses encode a gene with similarity to a retroviral superantigen gene (sag) of the unrelated mouse mammary tumour virus (MMTV). We uncover ancient retroviral sags in over 20 mammals to reconstruct their shared history with herpesviral sags, revealing that the acquisition is a convergent evolutionary event. A retrovirus circulating in South American primates over 10 million years ago was the source of sag in two monkey herpesviruses, and a different retrovirus was the source of sag in a Peruvian rodent herpesvirus. We further show through a timescaled phylogenetic analysis that a cross-species transmission of monkey herpesviruses occurred after the acquisition of sag. These results reveal that a diverse range of ancient sag-containing retroviruses independently donated sag twice from two separate lineages that are distinct from MMTV.

Herpesvirus saimiri MicroRNAs Preferentially Target Host Cell Cycle Regulators.

UNLABELLED: In latently infected marmoset T cells, Herpesvirus saimiri (HVS) expresses six microRNAs (known as miR-HSURs [H. saimiri U-rich RNAs]). The viral miR-HSURs are processed from chimeric primary transcripts, each containing a noncoding U-rich RNA (HSUR) and a pre-miRNA hairpin. To uncover the functions of miR-HSURs, we identified mRNA targets in infected cells using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP). HITS-CLIP revealed hundreds of robust Argonaute (Ago) binding sites mediated by miR-HSURs that map to the host genome but few in the HVS genome. Gene ontology analysis showed that several pathways regulating the cell cycle are enriched among cellular targets of miR-HSURs. Interestingly, miR-HSUR4-3p represses expression of the p300 transcriptional coactivator by binding the open reading frame of its mRNA. miR-HSUR5-3p directly regulates BiP, an endoplasmic reticulum (ER)-localized chaperone facilitating maturation of major histocompatibility complex class I (MHC-I) and the antiviral response. miR-HSUR5-3p also robustly downregulates WEE1, a key negative regulator of cell cycle progression, leading to reduced phosphorylation of its substrate, cyclin-dependent kinase (Cdk1). Consistently, inhibition of miR-HSUR5-3p in HVS-infected cells decreases their proliferation. Together, our results shed light on the roles of viral miRNAs in cellular transformation and viral latency. IMPORTANCE: Viruses express miRNAs during various stages of infection, suggesting that viral miRNAs play critical roles in the viral life cycle. Compared to protein-coding genes, the functions of viral miRNAs are not well understood. This is because it has been challenging to identify their mRNA targets. Here, we focused on the functions of the recently discovered HVS miRNAs, called miR-HSURs. HVS is an oncogenic gammaherpesvirus that causes acute T-cell lymphomas and leukemias in New World primates and transforms human T cells. A better understanding of HVS biology will help advance our knowledge of virus-induced oncogenesis. Because numerous cellular miRNAs play crucial roles in cancer, viral miRNAs from the highly oncogenic HVS might also be important for transformation. Here, we found that the miR-HSURs preferentially modulate expression of host cell cycle regulators, as well as antiviral response factors. Our work provides further insight into the functions of herpesviral miRNAs in virus-induced oncogenesis and latency.

The host Integrator complex acts in transcription-independent maturation of herpesvirus microRNA 3' ends.

Herpesvirus saimiri (HVS) is an oncogenic γ-herpesvirus that produces microRNAs (miRNAs) by cotranscription of precursor miRNA (pre-miRNA) hairpins immediately downstream from viral small nuclear RNAs (snRNA). The host cell Integrator complex, which recognizes the snRNA 3' end processing signal (3' box), generates the 5' ends of HVS pre-miRNA hairpins. Here, we identify a novel 3' box-like sequence (miRNA 3' box) downstream from HVS pre-miRNAs that is essential for miRNA biogenesis. In vivo knockdown and rescue experiments confirmed that the 3' end processing of HVS pre-miRNAs also depends on Integrator activity. Interaction between Integrator and HVS primary miRNA (pri-miRNA) substrates that contain only the miRNA 3' box was confirmed by coimmunoprecipitation and an in situ proximity ligation assay (PLA) that we developed to localize specific transient RNA-protein interactions inside cells. Surprisingly, in contrast to snRNA 3' end processing, HVS pre-miRNA 3' end processing by Integrator can be uncoupled from transcription, enabling new approaches to study Integrator enzymology.

Alternative capture of noncoding RNAs or protein-coding genes by herpesviruses to alter host T cell function.

In marmoset T cells transformed by Herpesvirus saimiri (HVS), a viral U-rich noncoding (nc) RNA, HSUR 1, specifically mediates degradation of host microRNA-27 (miR-27). High-throughput sequencing of RNA after crosslinking immunoprecipitation (HITS-CLIP) identified mRNAs targeted by miR-27 as enriched in the T cell receptor (TCR) signaling pathway, including GRB2. Accordingly, transfection of miR-27 into human T cells attenuates TCR-induced activation of mitogen-activated protein kinases (MAPKs) and induction of CD69. MiR-27 also robustly regulates SEMA7A and IFN-γ, key modulators and effectors of T cell function. Knockdown or ectopic expression of HSUR 1 alters levels of these proteins in virally transformed cells. Two other T-lymphotropic γ-herpesviruses, AlHV-1 and OvHV-2, do not produce a noncoding RNA to downregulate miR-27 but instead encode homologs of miR-27 target genes. Thus, oncogenic γ-herpesviruses have evolved diverse strategies to converge on common targets in host T cells.

Competitive and cooperative interactions mediate RNA transfer from herpesvirus saimiri ORF57 to the mammalian export adaptor ALYREF.

The essential herpesvirus adaptor protein HVS ORF57, which has homologs in all other herpesviruses, promotes viral mRNA export by utilizing the cellular mRNA export machinery. ORF57 protein specifically recognizes viral mRNA transcripts, and binds to proteins of the cellular transcription-export (TREX) complex, in particular ALYREF. This interaction introduces viral mRNA to the NXF1 pathway, subsequently directing it to the nuclear pore for export to the cytoplasm. Here we have used a range of techniques to reveal the sites for direct contact between RNA and ORF57 in the absence and presence of ALYREF. A binding site within ORF57 was characterized which recognizes specific viral mRNA motifs. When ALYREF is present, part of this ORF57 RNA binding site, composed of an α-helix, binds preferentially to ALYREF. This competitively displaces viral RNA from the α-helix, but contact with RNA is still maintained by a flanking region. At the same time, the flexible N-terminal domain of ALYREF comes into contact with the viral RNA, which becomes engaged in an extensive network of synergistic interactions with both ALYREF and ORF57. Transfer of RNA to ALYREF in the ternary complex, and involvement of individual ORF57 residues in RNA recognition, were confirmed by UV cross-linking and mutagenesis. The atomic-resolution structure of the ORF57-ALYREF interface was determined, which noticeably differed from the homologous ICP27-ALYREF structure. Together, the data provides the first site-specific description of how viral mRNA is locked by a herpes viral adaptor protein in complex with cellular ALYREF, giving herpesvirus access to the cellular mRNA export machinery. The NMR strategy used may be more generally applicable to the study of fuzzy protein-protein-RNA complexes which involve flexible polypeptide regions.

Idiopathic pulmonary fibrosis is strongly associated with productive infection by herpesvirus saimiri.

Idiopathic pulmonary fibrosis is a fatal disease without effective therapy or diagnostic test. To investigate a potential role for γ-herpesviruses in this disease, 21 paraffin-embedded lung biopsies from patients diagnosed with idiopathic pulmonary fibrosis and 21 lung biopsies from age-matched controls with pulmonary fibrosis of known etiology were examined for a series of γ-herpesviruses' DNA/RNA and related proteins using in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR)-based methods. We detected four proteins known to be in the genome of several γ-herpesviruses (cyclin D, thymidylate synthase, dihydrofolate reductase, and interleukin-17) that were strongly co-expressed in the regenerating epithelial cells of each of the 21 idiopathic pulmonary fibrosis cases and not in the benign epithelia of the controls. Among the γ-herpesviruses, only herpesvirus saimiri expresses all four of these 'pirated' mammalian proteins. We found herpesvirus saimiri DNA in the regenerating epithelial cells of 21/21 idiopathic pulmonary fibrosis cases using four separate probe sets but not in the 21 controls. RT-PCR showed that the source of the cyclin D RNA in active idiopathic pulmonary fibrosis was herpesvirus saimiri and not human. We cloned and sequenced part of genome corresponding to the DNA polymerase herpesvirus saimiri gene from an idiopathic pulmonary fibrosis sample and it matched 100% with the published viral sequence. These data are consistent with idiopathic pulmonary fibrosis representing herpesvirus saimiri-induced pulmonary fibrosis. Thus, treatment directed against viral proliferation and/or viral-associated proteins may halt disease progression. Further, demonstration of the viral nucleic acids or proteins may help diagnose the disease.

The use of high-frequency ultrasound imaging and biofluorescence for in vivo evaluation of gene therapy vectors.

BACKGROUND: Non-invasive imaging of the biodistribution of novel therapeutics including gene therapy vectors in animal models is essential. METHODS: This study assessed the utility of high-frequency ultrasound (HF-US) combined with biofluoresence imaging (BFI) to determine the longitudinal impact of a Herpesvirus saimiri amplicon on human colorectal cancer xenograft growth. RESULTS: HF-US imaging of xenografts resulted in an accurate and informative xenograft volume in a longitudinal study. The volumes correlated better with final ex vivo volume than mechanical callipers (R2 = 0.7993, p = 0.0002 vs. R2 = 0.7867, p = 0.0014). HF-US showed that the amplicon caused lobe formation. BFI demonstrated retention and expression of the amplicon in the xenografts and quantitation of the fluorescence levels also correlated with tumour volumes. CONCLUSIONS: The use of multi-modal imaging provided useful and enhanced insights into the behaviour of gene therapy vectors in vivo in real-time. These relatively inexpensive technologies are easy to incorporate into pre-clinical studies.

Potential of herpesvirus saimiri-based vectors to reprogram a somatic Ewing's sarcoma family tumor cell line.

Herpesvirus saimiri (HVS) infects a range of human cell types with high efficiency. Upon infection, the viral genome can persist as high-copy-number, circular, nonintegrated episomes that segregate to progeny cells upon division. This allows HVS-based vectors to stably transduce a dividing cell population and provide sustained transgene expression in vitro and in vivo. Moreover, the HVS episome is able to persist and provide prolonged transgene expression during in vitro differentiation of mouse and human hemopoietic progenitor cells. Together, these properties are advantageous for induced pluripotent stem cell (iPSC) technology, whereby stem cell-like cells are generated from adult somatic cells by exogenous expression of specific reprogramming factors. Here we assess the potential of HVS-based vectors for the generation of induced pluripotent cancer stem-like cells (iPCs). We demonstrate that HVS-based exogenous delivery of Oct4, Nanog, and Lin28 can reprogram the Ewing's sarcoma family tumor cell line A673 to produce stem cell-like colonies that can grow under feeder-free stem cell culture conditions. Further analysis of the HVS-derived putative iPCs showed some degree of reprogramming into a stem cell-like state. Specifically, the putative iPCs had a number of embryonic stem cell characteristics, staining positive for alkaline phosphatase and SSEA4, in addition to expressing elevated levels of pluripotent marker genes involved in proliferation and self-renewal. However, differentiation trials suggest that although the HVS-derived putative iPCs are capable of differentiation toward the ectodermal lineage, they do not exhibit pluripotency. Therefore, they are hereby termed induced multipotent cancer cells.