RESUMEN

The nuclear lamina is a dense network of intermediate filaments beneath the inner nuclear membrane. Composed of A-type lamins (lamin A/C) and B-type lamins (lamins B1 and B2), the nuclear lamina provides a scaffold for the nuclear envelope and chromatin, thereby maintaining the structural integrity of the nucleus. A-type lamins are also found inside the nucleus where they interact with chromatin and participate in gene regulation. Viruses replicating in the cell nucleus have to overcome the nuclear envelope during the initial phase of infection and during the nuclear egress of viral progeny. Here, we focused on the role of lamins in the replication cycle of a dsDNA virus, mouse polyomavirus. We detected accumulation of the major capsid protein VP1 at the nuclear periphery, defects in nuclear lamina staining and different lamin A/C phosphorylation patterns in the late phase of mouse polyomavirus infection, but the nuclear envelope remained intact. An absence of lamin A/C did not affect the formation of replication complexes but did slow virus propagation. Based on our findings, we propose that the nuclear lamina is a scaffold for replication complex formation and that lamin A/C has a crucial role in the early phases of infection with mouse polyomavirus.

RESUMEN

Background: BK polyomavirus (BKPyV)-associated nephropathy (BKPyVAN) causes renal allograft dysfunction and graft loss. However, the mechanism of BKPyV replication after kidney transplantation is unclear. Clinical studies have demonstrated that immunosuppressants and renal ischemia-reperfusion injury (IRI) are risk factors for BKPyV infection. Studying the pathogenic mechanism of BKPyV is limited by the inability of BKPyV to infect the animal. Mouse polyomavirus (MPyV) is a close homolog of BKPyV. We used a model of MPyV infection to investigate the core genes and underlying mechanism of IRI and immunosuppressants to promote polyomavirus replication. Materials and Methods: One-day-old male C57BL/6 mice were intraperitoneally injected with MPyV. At week 9 post-infection, all mice were randomly divided into IRI, immunosuppressant, and control groups and treated accordingly. IRI was established by clamping the left renal pedicle. Subsequently, kidney specimens were collected for detecting MPyV DNA, histopathological observation, and high-throughput RNA sequencing. Weighted gene correlation network analysis (WGCNA), protein-protein interaction network analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to screen for core genes and common signaling pathways involved in promoting MPyV replication by IRI and immunosuppressants. Results: After primary infection, MPyV established persistent infection in kidneys and subsequently was significantly increased by IRI or immunosuppressant treatment individually. In the IRI group, viral loads peaked on day 3 in the left kidney, which were significantly higher than those in the right kidney and the control group. In the immunosuppressant group, viral loads in the left kidney were significantly increased on day 3, which were significantly higher than those in the control group. Protein-protein interaction network analysis and WGCNA screened complement C3, epidermal growth factor receptor (EGFR), and FN1 as core genes. Pathway enrichment analysis based on the IRI- or immunosuppressant-related genes selected by WGCNA indicated that the NF-κB signaling pathway was the main pathway involved in promoting MPyV replication. The core genes were further confirmed using published datasets GSE47199 and GSE75693 in human polyomavirus-associated nephropathy. Conclusions: Our study demonstrated that IRI and immunosuppressants promote polyomavirus replication through common molecular mechanisms. In future studies, knockdown or specific inhibition of C3, EGFR, FN1, and NF-κB signaling pathway will further validate their critical roles in promoting polyomavirus replication.

Asunto(s)

Virus BK , Trasplante de Riñón , Nefritis Intersticial , Infecciones por Polyomavirus , Poliomavirus , Daño por Reperfusión , Animales , Virus BK/fisiología , Receptores ErbB , Femenino , Humanos , Inmunosupresores/efectos adversos , Trasplante de Riñón/efectos adversos , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B , Nefritis Intersticial/complicaciones , Poliomavirus/genética , Daño por Reperfusión/tratamiento farmacológico

RESUMEN

The mechanism by which DNA viruses interact with different DNA sensors and their connection with the activation of interferon (IFN) type I pathway are poorly understood. We investigated the roles of protein 204 (p204) and cyclic guanosine-adenosine synthetase (cGAS) sensors during infection with mouse polyomavirus (MPyV). The phosphorylation of IFN regulatory factor 3 (IRF3) and the stimulator of IFN genes (STING) proteins and the upregulation of IFN beta (IFN-ß) and MX Dynamin Like GTPase 1 (MX-1) genes were detected at the time of replication of MPyV genomes in the nucleus. STING knockout abolished the IFN response. Infection with a mutant virus that exhibits defective nuclear entry via nucleopores and that accumulates in the cytoplasm confirmed that replication of viral genomes in the nucleus is required for IFN induction. The importance of both DNA sensors, p204 and cGAS, in MPyV-induced IFN response was demonstrated by downregulation of the IFN pathway observed in p204-knockdown and cGAS-knockout cells. Confocal microscopy revealed the colocalization of p204 with MPyV genomes in the nucleus. cGAS was found in the cytoplasm, colocalizing with viral DNA leaked from the nucleus and with DNA within micronucleus-like bodies, but also with the MPyV genomes in the nucleus. However, 2'3'-Cyclic guanosine monophosphate-adenosine monophosphate synthesized by cGAS was detected exclusively in the cytoplasm. Biochemical assays revealed no evidence of functional interaction between cGAS and p204 in the nucleus. Our results provide evidence for the complex interactions of MPyV and DNA sensors including the sensing of viral genomes in the nucleus by p204 and of leaked viral DNA and micronucleus-like bodies in the cytoplasm by cGAS.

Asunto(s)

ADN Viral/inmunología , Inmunidad Innata/inmunología , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Nucleotidiltransferasas/metabolismo , Fosfoproteínas/metabolismo , Infecciones por Polyomavirus/inmunología , Poliomavirus/inmunología , Infecciones Tumorales por Virus/inmunología , Animales , ADN Viral/genética , Interacciones Huésped-Patógeno , Interferón beta/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratones , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Nucleotidiltransferasas/antagonistas & inhibidores , Nucleotidiltransferasas/genética , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Fosforilación , Poliomavirus/genética , Infecciones por Polyomavirus/virología , Infecciones Tumorales por Virus/virología

RESUMEN

The tumorigenic potential of mouse polyomavirus (MPyV) has been studied for decades in cell culture models and has been mainly attributed to nonstructural middle T antigen (MT), which acts as a scaffold signal adaptor, activates Src tyrosine kinases, and possesses transforming ability. We hypothesized that MPyV could also transform mouse cells independent of MT via a Toll-like receptor 4 (TLR4)-mediated inflammatory mechanism. To this end, we investigated the interaction of MPyV with TLR4 in mouse embryonic fibroblasts (MEFs) and 3T6 cells, resulting in secretion of interleukin 6 (IL-6), independent of active viral replication. TLR4 colocalized with MPyV capsid protein VP1 in MEFs. Neither TLR4 activation nor recombinant IL-6 inhibited MPyV replication in MEFs and 3T6 cells. MPyV induced STAT3 phosphorylation through both direct and MT-dependent and indirect and TLR4/IL-6-dependent mechanisms. We demonstrate that uninfected mouse fibroblasts exposed to the cytokine environment from MPyV-infected fibroblasts upregulated the expressions of MCP-1, CCL-5, and α-SMA. Moreover, the cytokine microenvironment increased the invasiveness of MEFs and CT26 carcinoma cells. Collectively, TLR4 recognition of MPyV induces a cytokine environment that promotes the cancer-associated fibroblast (CAF)-like phenotype in noninfected fibroblasts and increases cell invasiveness.

RESUMEN

Members of the Polyomaviridae family differ in their host range, pathogenesis, and disease severity. To date, some of the most studied polyomaviruses include human JC, BK, and Merkel cell polyomavirus and non-human subspecies murine and simian virus 40 (SV40) polyomavirus. Although dichotomies in host range and pathogenesis exist, overlapping features of the infectious cycle illuminate the similarities within this virus family. Of particular interest to human health, JC, BK, and Merkel cell polyomavirus have all been linked to critical, often fatal, illnesses, emphasizing the importance of understanding the underlying viral infections that result in the onset of these diseases. As there are significant overlaps in the capacity of polyomaviruses to cause disease in their respective hosts, recent advancements in characterizing the infectious life cycle of non-human murine and SV40 polyomaviruses are key to understanding diseases caused by their human counterparts. This review focuses on the molecular mechanisms by which different polyomaviruses hijack cellular processes to attach to host cells, internalize, traffic within the cytoplasm, and disassemble within the endoplasmic reticulum (ER), prior to delivery to the nucleus for viral replication. Unraveling the fundamental processes that facilitate polyomavirus infection provides deeper insight into the conserved mechanisms of the infectious process shared within this virus family, while also highlighting critical unique viral features.

Asunto(s)

Interacciones Microbiota-Huesped/genética , Poliomavirus/genética , Internalización del Virus , Replicación Viral , Animales , Núcleo Celular/virología , Especificidad del Huésped , Humanos , Poliomavirus/patogenicidad , Infecciones por Polyomavirus/virología

RESUMEN

Viruses have evolved mechanisms to manipulate microtubules (MTs) for the efficient realization of their replication programs. Studying the mechanisms of replication of mouse polyomavirus (MPyV), we observed previously that in the late phase of infection, a considerable amount of the main structural protein, VP1, remains in the cytoplasm associated with hyperacetylated microtubules. VP1-microtubule interactions resulted in blocking the cell cycle in the G2/M phase. We are interested in the mechanism leading to microtubule hyperacetylation and stabilization and the roles of tubulin acetyltransferase 1 (αTAT1) and deacetylase histone deacetylase 6 (HDAC6) and VP1 in this mechanism. Therefore, HDAC6 inhibition assays, αTAT1 knock out cell infections, in situ cell fractionation, and confocal and TIRF microscopy were used. The experiments revealed that the direct interaction of isolated microtubules and VP1 results in MT stabilization and a restriction of their dynamics. VP1 leads to an increase in polymerized tubulin in cells, thus favoring αTAT1 activity. The acetylation status of MTs did not affect MPyV infection. However, the stabilization of MTs by VP1 in the late phase of infection may compensate for the previously described cytoskeleton destabilization by MPyV early gene products and is important for the observed inhibition of the G2→M transition of infected cells to prolong the S phase.

Asunto(s)

Acetiltransferasas/genética , Proteínas de la Cápside/genética , Interacciones Microbiota-Huesped , Microtúbulos/metabolismo , Poliomavirus/metabolismo , Acetilación , Acetiltransferasas/metabolismo , Animales , Proteínas de la Cápside/metabolismo , Ciclo Celular , Línea Celular , Citoplasma/metabolismo , Fibroblastos/virología , Histona Desacetilasa 6/genética , Histona Desacetilasa 6/metabolismo , Ratones , Microtúbulos/virología , Poliomavirus/genética , Procesamiento Proteico-Postraduccional , Tubulina (Proteína)/metabolismo

RESUMEN

Programmed cell death-1 (PD-1) receptor signaling dampens the functionality of T cells faced with repetitive antigenic stimulation from chronic infections or tumors. Using intracerebral (i.c.) inoculation with mouse polyomavirus (MuPyV), we have shown that CD8 T cells establish a PD-1hi, tissue-resident memory population in the brains (bTRM) of mice with a low-level persistent infection. In MuPyV encephalitis, PD-L1 was expressed on infiltrating myeloid cells, microglia and astrocytes, but not on oligodendrocytes. Engagement of PD-1 on anti-MuPyV CD8 T cells limited their effector activity. NanoString gene expression analysis showed that neuroinflammation was higher in PD-L1-/- than wild type mice at day 8 post-infection, the peak of the MuPyV-specific CD8 response. During the persistent phase of infection, however, the absence of PD-1 signaling was found to be associated with a lower inflammatory response than in wild type mice. Genetic disruption and intracerebroventricular blockade of PD-1 signaling resulted in an increase in number of MuPyV-specific CD8 bTRM and the fraction of these cells expressing CD103, the αE integrin commonly used to define tissue-resident T cells. However, PD-L1-/- mice persistently infected with MuPyV showed impaired virus control upon i.c. re-infection with MuPyV. Collectively, these data reveal a temporal duality in PD-1-mediated regulation of MuPyV-associated neuroinflammation. PD-1 signaling limited the severity of neuroinflammation during acute infection but sustained a level of inflammation during persistent infection for maintaining control of virus re-infection.

Asunto(s)

Encéfalo/inmunología , Linfocitos T CD8-positivos/inmunología , Encefalitis Viral/inmunología , Infecciones por Polyomavirus/inmunología , Poliomavirus/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Animales , Antígeno B7-H1/genética , Antígeno B7-H1/inmunología , Encéfalo/patología , Linfocitos T CD8-positivos/patología , Encefalitis Viral/genética , Encefalitis Viral/patología , Femenino , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Ratones , Ratones Noqueados , Infecciones por Polyomavirus/genética , Infecciones por Polyomavirus/patología , Receptor de Muerte Celular Programada 1/genética

RESUMEN

Opportunistic viruses are a major problem for immunosuppressed individuals, particularly following organ or stem cell transplantation. Current treatments are non-existent or suffer from problems such as high toxicity or development of resistant strains. We previously published that a trafficking inhibitor that targets a host protein greatly reduces the replication of human cytomegalovirus. This inhibitor was also shown to be moderately effective against polyomaviruses, another family of opportunistic viruses. We have developed a panel of analogues for this inhibitor and have shown that these analogues maintain their high efficacy against HCMV, while substantially lowering the concentration required to inhibit polyomavirus replication. By targeting a host protein these compounds are able to inhibit the replication of two very different viruses. These observations open up the possibility of pan-viral inhibitors for immunosuppressed individuals that are effective against multiple, diverse opportunistic viruses.

Asunto(s)

Antivirales/farmacología , Citomegalovirus/fisiología , Replicación Viral/efectos de los fármacos , Animales , Antivirales/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Humanos , Ratones , Poliomavirus/fisiología , Quinazolinonas/química , Quinazolinonas/farmacología

RESUMEN

The mechanism used by mouse polyomavirus (MPyV) overcomes the crowded cytosol to reach the nucleus has not been fully elucidated. Here, we investigated the involvement of importin α/ß1 mediated transport in the delivery of MPyV genomes into the nucleus. Interactions of the virus with importin ß1 were studied by co-immunoprecipitation and proximity ligation assay. For infectivity and nucleus delivery assays, the virus and its capsid proteins mutated in the nuclear localization signals (NLSs) were prepared and produced. We found that at early times post infection, virions bound importin ß1 in a time dependent manner with a peak of interactions at 6 h post infection. Mutation analysis revealed that only when the NLSs of both VP1 and VP2/3 were disrupted, virus did not bind efficiently to importin ß1 and its infectivity remarkably decreased (by 80%). Nuclear targeting of capsid proteins was improved when VP1 and VP2 were co-expressed. VP1 and VP2 were effectively delivered into the nucleus, even when one of the NLS, either VP1 or VP2, was disrupted. Altogether, our results showed that MPyV virions can use VP1 and/or VP2/VP3 NLSs in concert or individually to bind importins to deliver their genomes into the cell nucleus.

Asunto(s)

Proteínas de la Cápside/metabolismo , ADN Viral/metabolismo , Carioferinas/metabolismo , Infecciones por Polyomavirus/metabolismo , Infecciones por Polyomavirus/virología , Poliomavirus/fisiología , Sustitución de Aminoácidos , Animales , Transporte Biológico , Proteínas de la Cápside/genética , Línea Celular , Núcleo Celular , Técnica del Anticuerpo Fluorescente , Ratones , Mutación , Señales de Localización Nuclear/genética , Poliomavirus/ultraestructura , Unión Proteica , Ensamble de Virus

RESUMEN

Polyomaviruses (PyVs) silently infect most humans, but they can cause life-threatening diseases in immunocompromised individuals. The JC polyomavirus (JCPyV) induces progressive multifocal leukoencephalopathy, a severe demyelinating disease in multiple sclerosis patients receiving immunomodulatory therapy, and BK polyomavirus (BKPyV)-associated nephropathy is a major cause of kidney allograft failure. No effective anti-PyV agents are available. Several compounds have been reported to possess anti-PyV activity in vitro, but none have shown efficacy in clinical trials. Productive PyV infection involves usurping the cellular retrograde vesicular transport pathway to enable endocytosed virions to navigate to the endoplasmic reticulum where virion uncoating begins. Compounds inhibiting this pathway have been shown to reduce infection by simian virus 40 (SV40), JCPyV, and BKPyV in tissue culture. In this study, we investigated the potential of Retro-2.1, a retrograde transport inhibitor, to limit infection by mouse polyomavirus (MuPyV) in vivo. We found that Retro-2.1 significantly reduced MuPyV levels in the kidney during acute infection without affecting renal function or the MuPyV-specific CD8 T cell response. To approximate the clinical setting of PyV resurgence in immunocompromised hosts, we showed that antibody-mediated depletion of T cells in persistently infected mice elevated MuPyV levels in the kidney and that Retro-2.1 blunted this increase in virus levels. In summary, these data indicate that inhibition of retrograde vesicular transport in vivo controls infection in a natural PyV mouse model and supports development of these compounds as potential therapeutic agents for individuals at risk for human PyV-associated diseases. IMPORTANCE PyVs can cause significant morbidity and mortality in immunocompromised individuals. No clinically efficacious anti-PyV therapeutic agents are available. A recently identified inhibitor of retrograde transport, Retro-2cycl, blocks movement of PyV virion-containing vesicles from early endosomes to the endoplasmic reticulum, an early step in the PyV life cycle. Retro-2cycl and its derivatives have been shown to inhibit infection by human PyVs in tissue culture. Here, we demonstrate that a derivative of Retro-2cycl, Retro-2.1, reduces infection by MuPyV in the kidneys of acutely infected mice. Mimicking the common clinical scenario of PyV resurgence, we further show that MuPyV levels increase in the kidneys of immunocompromised, persistently infected mice and that this increase is inhibited by Retro-2.1. These data provide the first evidence for control of a natural PyV infection in vivo by administration of an inhibitor of retrograde transport.

RESUMEN

VP1, the major structural protein of the mouse polyomavirus (MPyV), is the major architectural component of the viral capsid. Its pentamers are able to self-assemble into capsid-like particles and to non-specifically bind DNA. Surface loops of the protein interact with sialic acid of ganglioside receptors. Although the replication cycle of the virus, including virion morphogenesis, proceeds in the cell nucleus, a substantial fraction of the protein is detected in the cytoplasm of late-phase MPyV-infected cells. In this work, we detected VP1 mainly in the cytoplasm of mammalian cells transfected with plasmid expressing VP1. In the cytoplasm, VP1-bound microtubules, including the mitotic spindle, and the interaction of VP1 with microtubules resulted in cell cycle block at the G2/M phase. Furthermore, in the late phase of MPyV infection and in cells expressing VP1, microtubules were found to be hyperacetylated. We then sought to understand how VP1 interacts with microtubules. Dynein is not responsible for the VP1-microtubule association, as neither overexpression of p53/dynamitin nor treatment with ciliobrevin-D (an inhibitor of dynein activity) prevented binding of VP1 to microtubules. A pull-down assay for VP1-interacting proteins identified the heat shock protein 90 (Hsp90) chaperone, and Hsp90 was also detected in the VP1-microtubule complexes. Although Hsp90 is known to be associated with acetylated microtubules, it does not mediate the interaction between VP1 and microtubules. Our study provides insight into the role of the major structural protein in MPyV replication, indicating that VP1 is a multifunctional protein that participates in the regulation of cell cycle progression in MPyV-infected cells.

Asunto(s)

Proteínas de la Cápside/metabolismo , Células Epiteliales/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Microtúbulos/metabolismo , Poliomavirus/metabolismo , Virión/metabolismo , Acetilación , Animales , Proteínas de la Cápside/genética , Núcleo Celular/metabolismo , Núcleo Celular/virología , Citoplasma/metabolismo , Citoplasma/virología , Células Epiteliales/virología , Femenino , Puntos de Control de la Fase G2 del Ciclo Celular , Expresión Génica , Células HEK293 , Proteínas HSP90 de Choque Térmico/genética , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/virología , Ratones , Microtúbulos/virología , Células 3T3 NIH , Plásmidos/química , Plásmidos/metabolismo , Poliomavirus/genética , Unión Proteica , Transfección , Virión/genética

RESUMEN

The proteins p150Sal2 (product of SALL2) and p53 share growth arrest and pro-apoptotic functions by independently inducing p21Cip1/Waf1 and BAX, and both proteins are targeted by the human papilloma virus E6 protein, leading to blockage of growth arrest in infected cells. Loss of both p53 and Sall2 in mice causes significantly higher mortality and metastasis rates compared with p53 single mutant mice. Therefore, p150Sal2 seems to have strong potential as a novel cancer biomarker for early diagnosis and risk prediction. Loss of SALL2 expression is observed in many cases of human serous ovarian carcinoma, whereas normal ovarian epithelial cells maintain high levels of the p150Sal2 protein, supporting an important tumor suppressive role for p150Sal2 in the human ovary. In contrast, p150Sal2 is a transcription factor required to convert differentiated glioblastoma cells into stem-like tumor-propagating cells, suggesting that its functional roles are dependent on tissue types and cellular context. The function of p150Sal2 in normal and diseased cells and possible therapeutic approaches are discussed in this review.

Asunto(s)

Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/genética , Carcinogénesis/genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Proteínas Oncogénicas Virales/metabolismo , Neoplasias Ováricas/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Animales , Apoptosis , Biomarcadores de Tumor/genética , Neoplasias Encefálicas/metabolismo , Carcinogénesis/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Proteínas de Unión al ADN , Femenino , Glioblastoma/metabolismo , Humanos , Ratones , Mutación , Neoplasias Ováricas/metabolismo , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/metabolismo

RESUMEN

Mouse polyomavirus (MPyV) is a member of the Polyomaviridae family, which comprises non-enveloped tumorigenic viruses infecting various vertebrates including humans and causing different pathogenic responses in the infected organisms. Despite the variations in host tropism and pathogenicity, the structure of the virions of these viruses is similar. The capsid, with icosahedral symmetry (ø, 45 nm, T = 7d), is composed of a shell of 72 capsomeres of structural proteins, arranged around the nucleocore containing approximately 5-kbp-long circular dsDNA in complex with cellular histones. MPyV has been one of the most studied polyomaviruses and serves as a model virus for studies of the mechanisms of cell transformation and virus trafficking, and for use in nanotechnology. It can be propagated in primary mouse cells (e.g., in whole mouse embryo cells) or in mouse epithelial or fibroblast cell lines. In this unit, propagation, purification, quantification, and storage of MPyV virions are presented.

Asunto(s)

Poliomavirus/crecimiento & desarrollo , Poliomavirus/aislamiento & purificación , Preservación Biológica/métodos , Carga Viral/métodos , Cultivo de Virus/métodos , Animales , Células Cultivadas , Ratones

RESUMEN

Some viruses and most eukaryotic cells have microRNAs that regulate the expression of many genes. Although many viral miRNAs have been identified, only a few have been included in in vivo functional studies. Here we show that a Py-encoded miRNA downregulates the expression of the pro-apoptotic factor Smad2, resulting in the suppression of the apoptosis pathway. To study the Py miRNA in an in vivo context, a miRNA-deficient mutant virus was created on the background of the LID virus strain which establishes a rapid and lethal infection in newborn mice. Apoptosis analysis on kidney tissues indicates that the pro-apoptotic pathway is targeted in the infected host as well. Suppression of apoptosis through targeting of Smad2 by the Py miRNA is expected to synergize with anti-apoptotic effects previously attributed to the polyoma tumor antigens in support of virus replication in the natural host.

Asunto(s)

Apoptosis/fisiología , Células Epiteliales/fisiología , Células Epiteliales/virología , MicroARNs/metabolismo , Poliomavirus/clasificación , Proteína Smad2/metabolismo , Animales , Línea Celular , Lentivirus , Ratones , MicroARNs/genética , Interferencia de ARN , Proteína Smad2/genética

RESUMEN

Mouse polyomavirus (MPyV) is considered a potential tool for the application of gene therapy; however, the current knowledge of the encapsulation of DNA into virions is vague. We used a series of assays based on the encapsidation of a reporter vector into MPyV pseudovirions to identify putative cis-acting elements that are involved in DNA encapsidation. None of the sequences that were derived from MPyV have been shown to solely enhance the encapsidation of a reporter vector in the assay. The frequency of encapsidation strongly correlated with the total intracellular amount of the vector after transfection. The encapsidation of target DNA into the pseudovirions was shown to be non-specific, and the packaging of non-replicated DNA was observed. We propose that the actual concentration of target DNA at the sites of virion formation is the primary factor that determines its selection for encapsidation.

Asunto(s)

Cápside/metabolismo , Poliomavirus/fisiología , Virión/fisiología , Ensamble de Virus , Animales , Línea Celular , Genes Reporteros , Terapia Genética/instrumentación , Vectores Genéticos/genética , Vectores Genéticos/fisiología , Humanos , Ratones , Poliomavirus/genética , Virión/genética