High-throughput drug screen identifies calcium and calmodulin inhibitors that reduce JCPyV infection.

JC polyomavirus (JCPyV) is a nonenveloped, double-stranded DNA virus that infects the majority of the population. Immunocompetent individuals harbor infection in their kidneys, while severe immunosuppression can result in JCPyV spread to the brain, causing the neurodegenerative disease progressive multifocal leukoencephalopathy (PML). Due to a lack of approved therapies to treat JCPyV and PML, the disease results in rapid deterioration, and is often fatal. In order to identify potential antiviral treatments for JCPyV, a high-throughput, large-scale drug screen was performed using the National Institutes of Health Clinical Collection (NCC). Drugs from the NCC were tested for inhibitory effects on JCPyV infection, and drugs from various classes that reduced JCPyV infection were identified, including receptor agonists and antagonists, calcium signaling modulators, and enzyme inhibitors. Given the role of calcium signaling in viral infection including Merkel cell polyomavirus and simian virus 40 polyomavirus (SV40), calcium signaling inhibitors were further explored for the capacity to impact JCPyV infection. Calcium and calmodulin inhibitors trifluoperazine (TFP), W-7, tetrandrine, and nifedipine reduced JCPyV infection, and TFP specifically reduced viral internalization. Additionally, TFP and W-7 reduced infection by BK polyomavirus, SV40, and SARS-CoV-2. These results highlight specific inhibitors, some FDA-approved, for the possible treatment and prevention of JCPyV and several other viruses, and further illuminate the calcium and calmodulin pathway as a potential target for antiviral drug development.

Effect of ionic strength on the assembly of simian vacuolating virus capsid protein around poly(styrene sulfonate).

Virus-like particles (VLPs) are noninfectious nanocapsules that can be used for drug delivery or vaccine applications. VLPs can be assembled from virus capsid proteins around a condensing agent, such as RNA, DNA, or a charged polymer. Electrostatic interactions play an important role in the assembly reaction. VLPs assemble from many copies of capsid protein, with a combinatorial number of intermediates. Hence, the mechanism of the reaction is poorly understood. In this paper, we combined solution small-angle X-ray scattering (SAXS), cryo-transmission electron microscopy (TEM), and computational modeling to determine the effect of ionic strength on the assembly of Simian Vacuolating Virus 40 (SV40)-like particles. We mixed poly(styrene sulfonate) with SV40 capsid protein pentamers at different ionic strengths. We then characterized the assembly product by SAXS and cryo-TEM. To analyze the data, we performed Langevin dynamics simulations using a coarse-grained model that revealed incomplete, asymmetric VLP structures consistent with the experimental data. We found that close to physiological ionic strength, [Formula: see text] VLPs coexisted with VP1 pentamers. At lower or higher ionic strengths, incomplete particles coexisted with pentamers and [Formula: see text] particles. Including the simulated structures was essential to explain the SAXS data in a manner that is consistent with the cryo-TEM images.

Establishment of SV40 Large T-Antigen-Immortalized Yak Rumen Fibroblast Cell Line and the Fibroblast Responses to Lipopolysaccharide.

The yak lives in harsh alpine environments and the rumen plays a crucial role in the digestive system. Rumen-associated cells have unique adaptations and functions. The yak rumen fibroblast cell line (SV40T-YFB) was immortalized by introducing simian virus 40 large T antigen (SV40T) by lentivirus-mediated transfection. Further, we have reported the effects of lipopolysaccharide (LPS) of different concentrations on cell proliferation, extracellular matrix (ECM), and proinflammatory mediators in SV40T-YFB. The results showed that the immortalized yak rumen fibroblast cell lines were identified as fibroblasts that presented oval nuclei, a fusiform shape, and positive vimentin and SV40T staining after stable passage. Chromosome karyotype analysis showed diploid characteristics of yak (n = 60). LPS at different concentrations inhibited cell viability in a dose-dependent manner. SV40T-YFB treated with LPS increased mRNA expression levels of matrix metalloproteinases (MMP-2 and MMP-9), inflammatory cytokines (TNF-α, IL-1ß, IL-6), and urokinase-type plasminogen activator system components (uPA, uPAR). LPS inhibits the expression of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2), plasminogen activator inhibitor-2 (PAI-2), fibronectin (FN), anti-inflammatory factor IL-10, and collagen I (COL I) in SV40T-YFB. Overall, these results suggest that LPS inhibits cell proliferation and induces ECM degradation and inflammatory response in SV40T-YFB.

Characterization of a Human Gastrointestinal Stromal Tumor Cell Line Established by SV40LT-Mediated Immortalization.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the digestive tract and originate from the interstitial cells of Cajal (ICC), which is the pacemaker for peristaltic movement in the gastrointestinal tract. Existing GIST cell lines are widely used as cell models for in vitro experimental studies because the mutation sites are known. However, the immortalization methods of these cell lines are unknown, and no Chinese patient-derived GIST cell lines have been documented. Here, we transfected simian virus 40 large T antigen (SV40LT) into primary GIST cells to establish an immortalized human GIST cell line (ImGIST) for the first time. The ImGIST cells had neuronal cell-like irregular radioactive growth and retained the fusion growth characteristics of GIST cells. They stably expressed signature proteins, maintained the biological and genomic characteristics of normal primary GIST cells, and responded well to imatinib, suggesting that ImGIST could be a potential in vitro model for research in GIST to explore the molecular pathogenesis, drug resistance mechanisms, and the development of new adjuvant therapeutic options.

Unlicensed origin DNA melting by MCV and SV40 polyomavirus LT proteins is independent of ATP-dependent helicase activity.

Cellular eukaryotic replication initiation helicases are first loaded as head-to-head double hexamers on double-stranded (ds) DNA origins and then initiate S-phase DNA melting during licensed (once per cell cycle) replication. Merkel cell polyomavirus (MCV) large T (LT) helicase oncoprotein similarly binds and melts its own 98-bp origin but replicates multiple times in a single cell cycle. To examine the actions of this unlicensed viral helicase, we quantitated multimerization of MCV LT molecules as they assembled on MCV DNA origins using real-time single-molecule microscopy. MCV LT formed highly stable double hexamers having 17-fold longer mean lifetime (τ, >1,500 s) on DNA than single hexamers. Unexpectedly, partial MCV LT assembly without double-hexamer formation was sufficient to melt origin dsDNA as measured by RAD51, RPA70, or S1 nuclease cobinding. DNA melting also occurred with truncated MCV LT proteins lacking the helicase domain, but was lost from a protein without the multimerization domain that could bind only as a monomer to DNA. SV40 polyomavirus LT also multimerized to the MCV origin without forming a functional hexamer but still melted origin DNA. MCV origin melting did not require ATP hydrolysis and occurred for both MCV and SV40 LT proteins using the nonhydrolyzable ATP analog, adenylyl-imidodiphosphate (AMP-PNP). LT double hexamers formed in AMP-PNP, and melted DNA, consistent with direct LT hexamer assembly around single-stranded (ss) DNA without the energy-dependent dsDNA-to-ssDNA melting and remodeling steps used by cellular helicases. These results indicate that LT multimerization rather than helicase activity is required for origin DNA melting during unlicensed virus replication.

Immunogenic Properties and Antigenic Similarity of Virus-like Particles Derived from Human Polyomaviruses.

Polyomaviruses (PyVs) are highly prevalent in humans and animals. PyVs cause mild illness, however, they can also elicit severe diseases. Some PyVs are potentially zoonotic, such as simian virus 40 (SV40). However, data are still lacking about their biology, infectivity, and host interaction with different PyVs. We investigated the immunogenic properties of virus-like particles (VLPs) derived from viral protein 1 (VP1) of human PyVs. We immunised mice with recombinant HPyV VP1 VLPs mimicking the structure of viruses and compared their immunogenicity and cross-reactivity of antisera using a broad spectrum of VP1 VLPs derived from the PyVs of humans and animals. We demonstrated a strong immunogenicity of studied VLPs and a high degree of antigenic similarity between VP1 VLPs of different PyVs. PyV-specific monoclonal antibodies were generated and applied for investigation of VLPs phagocytosis. This study demonstrated that HPyV VLPs are highly immunogenic and interact with phagocytes. Data on the cross-reactivity of VP1 VLP-specific antisera revealed antigenic similarities among VP1 VLPs of particular human and animal PyVs and suggested possible cross-immunity. As the VP1 capsid protein is the major viral antigen involved in virus-host interaction, an approach based on the use of recombinant VLPs is relevant for studying PyV biology regarding PyV interaction with the host immune system.

A novel hybrid promoter capable of continuously producing proteins in high yield.

The establishment of cell lines with a high protein production is the most crucial objective in the field of biopharmaceuticals. To this end, efforts have been made to increase transgene expression through promoter improvement, but the efficiency or stability of protein production was insufficient for use in commercial production. Here, we developed a novel strategy to increase the efficiency and stability of protein production by hybridizing a promoter that exhibits higher expression levels at the transient level with a promoter that exhibits higher stability at the stable level. Expression levels of transgenes by each promoter were measured at transient and stable levels for five single promoters: Rous sarcoma virus (RSV), cytomegalovirus (CMV), human phosphoglycerate kinase (hPGK), simian virus 40 (SV40), and zebrafish ubiquitin B (Ubb). The hPGK promoter enabled high-yield transgene expression at transient levels and the SV40 promoter enabled sustained expression at stable levels. Therefore, hPGK and SV40 promoters were selected as candidates for establishing hybrid promoters and two hybrid promoters were constructed; one hybrid promoter in which the SV40 promoter is added before the hPGK promoter (a.k.a. SKYI) and the other hybrid promoter in which the SV40 promoter is added after the hPGK promoter (a.k.a. SKYII). Of the two hybrid promoters, the hybrid promoter SKYII promoted high-yield transgene expression at both transient and stable levels compared to single hPGK and SV40. Together, our findings open new doors in the field of biopharmaceuticals by presenting a novel promoter platform that can be used for high-yield and sustained protein production.

Bovine Polyomavirus-1 (Epsilonpolyomavirus bovis): An Emerging Fetal Pathogen of Cattle That Causes Renal Lesions Resembling Polyomavirus-Associated Nephropathy of Humans.

Bovine polyomavirus-1 (BoPyV-1, Epsilonpolyomavirus bovis) is widespread in cattle and has been detected in commercialized beef at supermarkets in the USA and Germany. BoPyV-1 has been questioned as a probable zoonotic agent with documented increase in seropositivity in people exposed to cattle. However, to date, BoPyV-1 has not been causally associated with pathology or disease in any animal species, including humans. Here we describe and illustrate pathological findings in an aborted bovine fetus naturally infected with BoPyV-1, providing evidence of its pathogenicity and probable abortigenic potential. Our results indicate that: (i) BoPyV-1 can cause severe kidney lesions in cattle, including tubulointerstitial nephritis with cytopathic changes and necrosis in tubular epithelial cells, tubular and interstitial inflammation, and interstitial fibroplasia; (ii) lesions are at least partly attributable to active viral replication in renal tubular epithelial cells, which have abundant intranuclear viral inclusions; (iii) BoPyV-1 large T (LT) antigen, resulting from early viral gene expression, can be detected in infected renal tubular epithelial cells using a monoclonal antibody raised against Simian Virus-40 polyomavirus LT antigen; and (iv) there is productive BoPyV-1 replication and virion assembly in the nuclei of renal tubular epithelial cells, as demonstrated by the ultrastructural observation of abundant arrays of viral particles with typical polyomavirus morphology. Altogether, these lesions resemble the "cytopathic-inflammatory pathology pattern" proposed in the pathogenesis of Human polyomavirus-1-associated nephropathy in immunocompromised people and kidney allograft recipients. Additionally, we sequenced the complete genome of the BoPyV-1 infecting the fetus, which represents the first whole genome of a BoPyV-1 from the Southern Hemisphere. Lastly, the BoPyV-1 strain infecting this fetus was isolated, causing a cytopathic effect in Madin-Darby bovine kidney cells. We conclude that BoPyV-1 is pathogenic to the bovine fetus under natural circumstances. Further insights into the epidemiology, biology, clinical relevance, and zoonotic potential of BoPyV-1 are needed.

Packaging of DNA origami in viral capsids: towards synthetic viruses.

We report a new type of nanoparticle, consisting of a nucleic acid core (>7500 nt) folded into a 35 nm DNA origami sphere, encapsulated by a capsid composed of all three SV40 virus capsid proteins. Compared to the prototype reported previously, whose capsid consists of VP1 only, the new nanoparticle closely adopts the unique intracellular pathway of the native SV40, suggesting that the proteins of the synthetic capsid retain their native viral functionality. Some of the challenges in the design of such near-future composite drugs destined for gene delivery are discussed.

Simian virus 40 DNA in immunocompetent children with respiratory disease.

Evidence of Simian virus 40 (SV40) DNA sequences or gene products has been reported in a variety of organ systems in humans. However, the route of transmission and the significance of SV40 polyomavirus infection in human are unknown. The aim of study was to characterize the frequency of SV40 infection in immunocompetent and immunocompromised patients with respiratory diseases. Respiratory specimens from patients with respiratory tract illness obtained from nasopharyngeal aspirates (n = 280) were screened for SV40 polyomavirus using real-time PCR; coinfection with other viruses was examined. Positive results were confirmed with sequencing. Of the 280 samples analysed, 2 (0.71%) were positive for SV40. SV40 was identified in nasopharyngeal aspirate samples from children aged 8 and 14 months who were immunocompetent. Both patients had upper or lower respiratory tract infection. Coinfections with other viruses were found in 50% of the SV40 positive samples. The data suggest that SV40 can infect respiratory tract, that respiratory tract may represent a route of transmission or a site for virus persistence, and that with the high rate of co-infection, SV40 may not involved in respiratory diseases.

Early in an SV40 infection, histone modifications correlate with the presence or absence of RNAPII and direction of transcription.

Since epigenetic regulation seemed likely to be involved in SV40 early transcription following infection, we have analyzed the organization of nucleosomes carrying histone modifications (acetyl-H3, acetyl-H4, H3K9me1, H3K9me3, H3K4me1, H3K4me3, H3K27me3, H4K20me1) at 30 min and 2 h post infection in SV40 minichromosomes prepared in the absence or presence of the transcription inhibitor dichloro-1-beta-d-ribofuranosyl benzimidazole. The former condition was used to determine how SV40 chromatin structure changed during early transcription, and the latter was used to determine the role of active transcription. The location of RNAPII was used as a marker to identify where histone modifications were most likely to be involved in regulation. Acetyl-H3 acted like epigenetic memory by being present at sites subsequently bound by RNAPII, while H3K9me1 and H3K27me3 were reorganized to the late side of the SV40 regulatory region apparently to repress late transcription. The organization of acetyl-H3 and H3K9me1 but not H3K27me3 required active transcription.

Long-read sequencing reveals complex patterns of wraparound transcription in polyomaviruses.

Polyomaviruses (PyV) are ubiquitous pathogens that can cause devastating human diseases. Due to the small size of their genomes, PyV utilize complex patterns of RNA splicing to maximize their coding capacity. Despite the importance of PyV to human disease, their transcriptome architecture is poorly characterized. Here, we compare short- and long-read RNA sequencing data from eight human and non-human PyV. We provide a detailed transcriptome atlas for BK polyomavirus (BKPyV), an important human pathogen, and the prototype PyV, simian virus 40 (SV40). We identify pervasive wraparound transcription in PyV, wherein transcription runs through the polyA site and circles the genome multiple times. Comparative analyses identify novel, conserved transcripts that increase PyV coding capacity. One of these conserved transcripts encodes superT, a T antigen containing two RB-binding LxCxE motifs. We find that superT-encoding transcripts are abundant in PyV-associated human cancers. Together, we show that comparative transcriptomic approaches can greatly expand known transcript and coding capacity in one of the simplest and most well-studied viral families.

Molecular dissection on inhibition of Ras-induced cellular senescence by small t antigen of SV40.

Simian virus 40 (SV40) is a potentially oncogenic virus of monkey origin. Transmission, prevalence, and pathogenicity rates of SV40 are unclear, but infection can occur in humans, for example individuals with high contact with rhesus macaques and individuals that received contaminated early batches of polio vaccines in 1950-1963. In addition, several human polyomaviruses, proven carcinogenic, are also highly common in global populations. Cellular senescence is a major mechanism of cancer prevention in vivo. Hyperactivation of Ras usually induces cellular senescence rather than cell transformation. Previous studies suggest small t antigen (ST) of SV40 may interfere with cellular senescence induced by Ras. In the current study, ST was demonstrated to inhibit Ras-induced cellular senescence (RIS) and accumulation of DNA damage in Ras-activated cells. In addition, ST suppressed the signal transmission from BRaf to MEK and thus blocked the downstream transmission of the activated Ras signal. B56γ knockdown mimicked the inhibitory effects of ST overexpression on RIS. Furthermore, KSR1 knockdown inhibited Ras activation and the subsequent cellular senescence. Further mechanism studies indicated that the phosphorylation level of KSR1 rather than the levels of the total protein regulates the activation of Ras signaling pathway. In sum, ST inhibits the continuous hyperactivation of Ras signals by interfering with the normal functions of PP2A-B56γ of dephosphorylating KSR1, thus inhibiting the occurrence of cellular senescence. Although the roles of SV40 in human carcinogenesis are controversial so far, our study has shown that ST of polyomaviruses has tumorigenic potential by inhibiting oncogene-induced senescence (OIS) as a proof of concept.

SV40 T antigen helicase domain regions responsible for oligomerisation regulate Okazaki fragment synthesis initiation.

The initiation of Okazaki fragment synthesis during cellular DNA replication is a crucial step for lagging strand synthesis, which is carried out by the primase function of DNA polymerase α-primase (Pol-prim). Since cellular replication protein A (RPA) prevents primase from starting RNA synthesis on single-stranded DNA (ssDNA), primase requires auxiliary factors, such as the simian virus 40 (SV40) T antigen (Tag), for the initiation reaction on RPA-bound ssDNA. Here, we investigated the ability of Tag variants and Tag protein complexes to bind to ssDNA and their resulting effects on the stimulation of Pol-prim on free and RPA-bound ssDNA. Atomic force microscopy imaging showed that while Tag131-627 (V350E/P417D) and Tag131-627 (L286D/R567E) (abbreviated as M1 and M2, respectively) could bind to ssDNA as monomers, these monomeric Tags could come together and bind to ssDNA as dimers as well. In a model assay for the initiation of Okazaki fragment synthesis, full-length Tag SV40 Tag1-708 and monomeric M2 stimulated DNA synthesis of Pol-prim on ssDNA and on RPA-bound ssDNA. In contrast, neither monomeric M1 nor M1-M2 dimers could stimulate Pol-prim, on ssDNA or on RPA-bound ssDNA. Overall, we show that a lack of stimulatory activity of monomeric M1 and M1-M2 dimers suggests that residues V350 and P417 are not only important for interactions between Tag molecules but also for protein-protein interactions within Okazaki fragment initiation complexes. Thus, we highlight that mutations in M1 are dominant negative with regard to Okazaki fragment initiation.

Interactions of large T-Antigen (LT) protein of polyomaviruses with p53 unfold their cancerogenic potential.

Polyomaviruses such as Simian Virus 40 (SV40) and John Cunningham Virus (JCV) have been extensively studied for their potential role in aiding oncogenic transformation. One of the mechanisms through which they do this is by inactivating p53, a known tumor suppressor, through one of their viral proteins, large T-antigen (LT). However, these two viruses represent only a fraction of existing polyomaviruses. Using Clustal Omega, we aligned the protein sequences of LT for 12 different polyomaviruses and found high similarity across polyomavirus LT. We then utilized Molecular Operating Environment (MOE) v2019.01 to compare the binding of SV40 LT to p53 and p53 to DNA to more precisely define the mechanism with which SV40 LT inactivates p53. By binding to p53 residues essential to DNA binding, SV40 LT prevents the proper interaction of p53 with DNA and consequently its fulfillment of transcription factor functions. To further explore the possibility for other polyomavirus LT to do the same, we either retrieved existing 3D structures from RCSB Protein Data Bank or generated 3D homology models of other polyomavirus LT and modeled their interactions with p53. These models interacted with p53 in a similar manner as SV40 LT and provide further evidence of the potential of other polyomavirus LT to inactivate p53. This work demonstrates the importance of investigating the oncogenic potential of polyomaviruses and elucidates future targets for cancer treatment.Communicated by Ramaswamy H. Sarma.

Lunapark-dependent formation of a virus-induced ER exit site contains multi-tubular ER junctions that promote viral ER-to-cytosol escape.

Viruses rearrange host membranes to support different entry steps. Polyomavirus simian virus 40 (SV40) reorganizes the endoplasmic reticulum (ER) membrane to generate focus structures that enable virus ER-to-cytosol escape, a decisive infection step. The molecular architecture of the ER exit site that might illuminate why it is ideally suited for membrane penetration is unknown. Here 3D focused ion beam scanning electron microscopy (FIB-SEM) reconstruction reveals that the ER focus structure consists of multi-tubular ER junctions where SV40 preferentially localizes, suggesting that tubular branch points are virus ER-to-cytosol penetration sites. Functional analysis demonstrates that lunapark-an ER membrane protein that typically stabilizes three-way ER junctions-relocates to the ER foci, where it supports focus formation, leading to SV40 ER escape and infection. Our results reveal how a virus repurposes the activity of an ER membrane protein to form a virus-induced ER substructure required for membrane escape and suggest that ER tubular junctions are vulnerable sites exploited by viruses for membrane penetration.

Inactivation of Polyomavirus SV40 as Surrogate for Human Papillomaviruses by Chemical Disinfectants.

Human papillomaviruses (HPV) are non-enveloped DNA viruses infecting cutaneous and mucosal squamous epithelia. Sexually transmitted HPV-types that are carcinogenic to humans such as HPV16 can induce cervical and other anogenital cancers. Virus transmission through fomites such as inadequately disinfected gynecological equipment is a further potential transmission route. Since HPV cannot be easily grown in cell culture, polyomavirus SV40 has been used as a surrogate virus when testing the virucidal activity of chemical disinfectants. So far, studies that have compared the virucidal activity of different disinfectants against HPV and SV40 are lacking. Here, we evaluated the susceptibility of HPV16 pseudovirus and SV40 to seven active biocidal substances using quantitative suspension tests. Ethanol, glutaraldehyde (GTA), dodecyldipropylentriamin (DPTA), and ortho-phthalaldehydes (OPA) were able to reduce the infectivity of HPV16 pseudovirus >99.99% after 5 min. In contrast, isopropanol, peracetic acid (PAA), and quaternary ammonium compounds with alkylamines (QAC) only led to a slight or no reduction in infectivity. Concerning SV40, only GTA (60 min contact time), PAA, and OPA had virus-inactivating effects. In conclusion, the virucidal activity of three out of seven disinfectants tested was different for HPV16 pseudovirus and SV40. In this study, SV40 was shown to be a reliable surrogate virus for HPV when testing isopropanol-, GTA-, QAC-, and OPA-based disinfectants.

Combining protein and RNA quantification to evaluate promoter activity by using dual-color fluorescent reporting systems.

Herein, Broccoli/mCherry and an EGFP/mCherry dual-color fluorescent reporting systems have been established to quantify the promoter activity at transcription and translation levels in eukaryotic cells. Based on those systems, four commonly used promoters (CMV and SV40 of Pol II and U6, H1 of Pol III) were accurately evaluated at both the transcriptional and translational levels by combining accurate protein and RNA quantification. Furthermore, we verified that Pol III promoters can induce proteins expression, and Pol II promoter can be applied to express RNA molecules with defined length by combining a self-cleaving ribozyme and an artificial poly(A) tail. The dual-color fluorescence reporting systems described here could play a significant role in evaluating other gene expression regulators for gene therapy.

Stem cells immortalized by hTERT perform differently from those immortalized by SV40LT in proliferation, differentiation, and reconstruction of matrix microenvironment.

Although matrix microenvironment has the potential to improve expanded stem cell proliferation and differentiation capacity, decellularized extracellular matrix (dECM) deposited by senescent cells does not contribute to the rejuvenation of adult stem cells, which has become a barrier to personalized stem cell therapy. Genetic modification is an effective strategy to protect cells from senescence but it carries the increased risk of malignant transformation and genetic instability. In this study, lentivirus carrying either human telomerase reverse transcriptase (hTERT) or simian virus 40 large T antigen (SV40LT) was used to transduce human infrapatellar fat pad-derived stem cells (IPFSCs). We found that virus transduction modified the proliferative, chondrogenic, and adipogenic abilities of IPFSCs. Interestingly, dECM deposited by immortalized cells significantly influenced replicative senescent IPFSCs in proliferation and differentiation preference, the effect of which is hinged on the approach of immortalization using either SV40LT or hTERT. Our findings indicate both dECM expansion and immortalization strategies can be used for replicative senescent adult stem cells' proliferation and lineage-specific differentiation, which benefits future stem cell-based tissue regeneration. This approach may also work for adult stem cells with premature senescence in elderly/aged patients, which needs further investigation. STATEMENT OF SIGNIFICANCE: Adult stem cells are a promising solution for autologous cell-based therapy. Unfortunately, cell senescence due to donor age and/or ex vivo expansion prevents clinical application. Recent progress with decellularized extracellular matrix provides a potential for the rejuvenation of senescent stem cells by improving their proliferation and differentiation capacities. Given the fact that the young matrix can provide a healthy and energetic microenvironment, in this study, two approaches using lentivirus transduction of hTERT and SV40LT were compared. The goal was to immortalize donor cells for deposition of decellularized extracellular matrix. The matrix was demonstrated to contribute diverging effects on the chondrogenic and adipogenic differentiation of expanded stem cells and exhibited proliferation benefits as well. These findings provide an invaluable asset for stem cell-based tissue regeneration.