Efficient propagation of progressive multifocal leukoencephalopathy-type JC virus in COS-7-derived cell lines stably expressing Tat protein of human immunodeficiency virus type 1

The high incidence of progressive multifocal leukoencephalopathy (PML) in AIDS patients compared with many other immunosuppressive diseases suggests that HIV-1 infection is strictly related to the activation of JC virus (JCV) propagation. In this report, propagation of PML-type JCV in COS-7-derived cell lines stably expressing HIV-1 Tat (COS-tat cells) has been examined. In COS-tat cells, production of viral particles and replication of genomic DNA were markedly increased compared to COS-7 cells, as judged by HA and real-time PCR analyses. These results demonstrate that COS-tat cells provide a useful model system for studying HIV-1 Tat-mediated propagation of PML-type JCV.     

Archetype JC Virus Efficiently Replicates in COS-7 Cells, Simian Cells Constitutively Expressing Simian Virus 40 T Antigen

JC polyomavirus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), is ubiquitous in humans, infecting children asymptomatically and then persisting in the kidney. Renal JCV is not latent but replicates to excrete progeny in the urine. The renal-urinary JCV DNAs carry the archetype regulatory region that generates various rearranged regulatory regions occurring in JCVs derived from the brains of PML patients. Tissue cultures that support the efficient growth of archetype JCV have not been reported. We studied whether archetype JCV could replicate in COS-7 cells, simian cells transformed with an origin-defective mutant of simian virus 40 (SV40). Efficient JCV replication, as detected by a hemagglutination assay, was observed in cultures transfected with five of the six archetype DNAs. The progeny JCVs could be passaged to fresh COS-7 cells. However, when the parental cells of COS-7 not expressing T antigen were transfected with archetype JCV DNAs, no viral replication was detected, indicating that SV40 T antigen is essential for the growth of JCV in COS-7 cells. The archetype regulatory region was conserved during viral growth in COS-7 cells, although a small proportion of JCV DNAs underwent rearrangements outside the regulatory region. We then attempted to recover archetype JCV from urine by viral culture in COS-7 cells. Efficient JCV production was observed in COS-7 cells infected with five of the six JCV-positive urine samples examined. Thus, COS-7 cells should be of use not only for the production of archetype JCV on a large scale but also for the isolation of archetype JCV from urine.     

Rearrangement of simian virus 40 regulatory region is not required for induction of progressive multifocal leukoencephalopathy in immunosuppressed rhesus monkeys

Rearrangements of the JC virus (JCV) regulatory region (RR) are consistently found in the brains of patients with progressive multifocal leukoencephalopathy (PML), whereas the archetype RR is present in their kidneys. In addition, the C terminus of the large T antigen (T-Ag) shows greater variability in PML than does the rest of the coding region. To determine whether similar changes in simian virus 40 (SV40) are necessary for disease induction in monkeys, we sequenced the SV40 RR and the C terminus of the T-Ag from the brain of simian/human immunodeficiency virus (SHIV)-infected monkey 18429, which presented spontaneously with an SV40-associated PML-like disease, as well as from the peripheral blood mononuclear cells (PBMC), kidneys, and brains of SV40-seronegative, SHIV-infected monkeys 21289 and 21306, which were inoculated with the 18429 brain SV40 isolate. These animals developed both SV40-associated PML and meningoencephalitis. Thirteen types of SV40 RR were characterized. Compared to the SV40 archetype, we identified RRs with variable deletions in either the origin of replication, the 21-bp repeat elements, or the late promoter, as well as deletions or duplications of the 72-bp enhancer. The archetype was the most prominent RR in the brain of monkey 18429. Shortly after inoculation, a wide range of RRs could be found in the PBMC of monkeys 21289 and 21306. However, the archetype RR became the predominant type in their blood, kidneys, and brains at the time of sacrifice. On the contrary, the T-Ag C termini remained identical in all compartments of the three animals. These results indicate that unlike JCV in humans, rearrangements of SV40 RR are not required for brain disease induction in immunosuppressed monkeys.     

JC virus DNA is present in many human brain samples from patients without progressive multifocal leukoencephalopathy.

Sections of normal and diseased brain and kidney tissues were screened for the presence of JC virus (JCV) DNA by using the polymerase chain reaction. As expected, all samples obtained from patients with progressive multifocal leukoencephalopathy (PML) tested positive when multiple JCV-specific primer and probe combinations were used. Unexpectedly, more than 50% of non-PML-affected brains were also found to harbor low levels of JCV DNA. To confirm that the positive signals seen in the tissue sections were not the result of contamination, amplified DNA was cloned and sequenced and in some cases was shown to represent strains of JCV not identified previously. Two predominant regulatory region configurations of JCV have been detected in the human host: archetype JCV, which is excreted in the urine of normal and immunocompromised individuals, and "PML-type" JCV found in diseased brains. This latter group of variants appears to derive from archetype JCV by the deletion and duplication of sequences within the promoter-enhancer region. In the present study, the archetype strain of JCV was identified only in normal kidney samples; JCV DNA found in non-PML-affected brain specimens and in kidney tissue from patients with PML resembled that of strains isolated from PML-affected brain tissue. Our findings indicate that JCV reaches the brain more frequently than previously thought and may persist at this site without causing demyelinating disease. A subsequent episode of prolonged immunodeficiency or a direct interaction with an immunocompromising agent (e.g., human immunodeficiency virus type 1) might activate the latent JCV infection and lead to the development of PML.     

SV40-transformed simian cells support the replication of early SV40 mutants

CV-1, an established line of simian cells permissive for lytic growth of SV40, were transformed by an origin-defective mutant of SV40 which codes for wild-type T antigen. Three transformed lines (COS-1, -3, -7) were established and found to contain T antigen; retain complete permissiveness for lytic growth of SV40; support the replication of tsA209 virus at 40 degrees C; and support the replication of pure populations of SV40 mutants with deletions in the early region. One of the lines (COS-1) contains a single integrated copy of the complete early region of SV40 DNA. These cells are possible hosts for the propagation of pure populations of recombinant SV40 viruses.     

Establishment of a temperature-inducible cell line for human plasminogen activator (tissue-type) by transfection of monkey cells with expression constructs

An expression construct for human tissue-type plasminogen activator (t-pA) cDNA [containing a simian virus 40 (SV40) origin of replication] was introduced into CV1, COS-7 and COSts2 cells; in the latter cell line the amount of functionally active large T antigen of SV40 is regulated by the temperature. In a transient system, the expression in COSts2 cells at the permissive temperature for large T antigen was improved sixfold compared to COS-7 cells. By cotransfection with a plasmid conferring resistance to G418 into COSts2 cells, a cell line (COSts2Glob t-pA) could be isolated with barely detectable expression of t-pA at the semi-permissive and non-permissive temperature and inducible secretion of t-pA at the permissive temperature. The kinetics of induction, inducibility after continued propagation at the semi-permissive temperature and the influence of the temperature during previous propagation on inducibility were investigated. The biological activity of the secreted material was demonstrated by a functional assay. Inducibility of t-pA by temperature was accompanied by a dramatic increase of the copy number of episomal plasmids (up to 2000 copies per cell).     

Rearranged JC Virus Noncoding Control Regions Found in Progressive Multifocal Leukoencephalopathy Patient Samples Increase Virus Early Gene Expression and Replication Rate

Polyomavirus JC (JCV) infects ∼60% of the general population, followed by asymptomatic urinary shedding in ∼20%. In patients with pronounced immunodeficiency, including HIV/AIDS, JCV can cause progressive multifocal leukoencephalopathy (PML), a devastating brain disease of high mortality. While JCV in the urine of healthy people has a linear noncoding control region called the archetype NCCR (at-NCCR), JCV in brain and cerebrospinal fluid (CSF) of PML patients bear rearranged NCCRs (rr-NCCRs). Although JCV NCCR rearrangements are deemed pathognomonic for PML, their role as a viral determinant is unclear. We sequenced JCV NCCRs found in CSF of eight HIV/AIDS patients newly diagnosed with PML and analyzed their effect on early and late gene expression using a bidirectional reporter vector recapitulating the circular polyomavirus early and late gene organization. The rr-NCCR sequences were highly diverse, but all increased viral early reporter gene expression in progenitor-derived astrocytes, glia-derived cells, and human kidney compared to the expression levels with the at-NCCR. The expression of simian virus 40 (SV40) large T antigen or HIV Tat expression in trans was associated with a strong increase of at-NCCR-controlled early gene expression, while rr-NCCRs were less responsive. The insertion of rr-NCCRs into the JCV genome backbone revealed higher viral replication rates for rr-NCCR compared to those of the at-NCCR JCV in human progenitor-derived astrocytes or glia cells, which was abrogated in SV40 large T-expressing COS-7 cells. We conclude that naturally occurring JCV rr-NCCR variants from PML patients confer increased early gene expression and higher replication rates compared to those of at-NCCR JCV and thereby increase cytopathology.Polyomavirus JC (JCV) infects approximately 60% of the general population, followed by asymptomatic urinary shedding in 20% of healthy individuals (20). Although JCV-associated nephropathy may occur in kidney transplant (14, 33) and HIV/AIDS patients (6, 27), the most prominent JCV disease is progressive multifocal leukoencephalopathy (PML) (44, 60). The pathology of PML was first described in 1958 as a rare complication of patients with chronic lymphocytic leukemia or Hodgkin''s lymphoma (3). Today, PML is recognized as a rare, virus-mediated demyelinating disease of the white brain matter in highly immunocompromised patients, including HIV/AIDS, transplantation, and chemotherapy patients and those exposed to immunomodulatory or depleting biologicals for the treatment of autoimmune diseases (29, 40). During the human immunodeficiency virus type 1 (HIV-1) pandemic, the incidence of PML rose significantly to rates of 1 to 8% prior to the use of highly active antiretroviral therapy (2, 5, 34). The definitive diagnosis requires brain tissue, but the detection of JCV by PCR in cerebrospinal fluid (CSF) is generally accepted for a laboratory-confirmed diagnosis in immunocompromised patients with (multi-)focal neurological deficits and corresponding radiological findings (8, 26). Due to the lack of effective antiviral therapy (13), the treatment of PML is based on improving overall immune functions. While this is difficult to achieve in cancer, chemotherapy, and transplantation, prompt antiretroviral therapy in HIV/AIDS patients has significantly improved PML survival, with increasing JCV-specific immune responses and declining intracerebral JCV replication (7, 15, 23, 35, 37). In patients diagnosed with PML after treatment with natalizumab for multiple sclerosis or inflammatory bowel disease, the removal of the monoclonal antibody by plasmapheresis has been tried to restore lymphocyte homing to, and the immune surveillance of, JCV replication sites in the central nervous system (38, 40, 52). However, the success of immune reconstitution in HIV/AIDS- and natalizumab-associated PML cases is limited by the fact that PML is typically diagnosed clinically by neurological deficits resulting from significant brain damage, where mounting antiviral immunity often may be too slow to modify the outcome. On the other hand, rapid recovery may cause immune reconstitution inflammatory syndrome with paradoxical clinical worsening and fatal outcomes (9, 16, 25, 38, 46). Although the etiologic role of JCV in PML is well documented, the pathogenesis and, in particular, the role of viral determinants is less clear. Virtually all JCV strains isolated from the brain or CSF of PML patients are characterized by highly variable genomic rearrangements of the noncoding control region (NCCR), which governs viral early and late genes in opposite directions of the circular polyomavirus DNA genome (1, 4, 31, 39, 41, 43, 49, 54, 59). In contrast, JCV detected in the urine of immunocompetent individuals show a consistent linear architecture called the archetype NCCR (at-NCCR). Thus, detecting rearranged NCCRs (rr-NCCRs) JCV in the central nervous system has been viewed as being derived from the archetype and closely linked to PML (4), but the functional consequences of rearrangements are unclear. To address the consequences of the rr-NCCR for JCV gene expression and replication, we characterized the sequences of JCV rr-NCCR from patients with PML and analyzed their effect on viral gene expression and replication with JCV at-NCCR in a bidirectional reporter assay and in recombinant JCV.     

Molecular biology and immunoregulation of human neurotropic JC virus in CNS

The human polyomavirus, JC virus (JCV), provides an excellent model system to investigate the reciprocal interaction of the immune and nervous systems. Infection with JCV occurs during childhood and the virus remains in the latent state with no apparent clinical symptoms. However, under immunosuppressed conditions, the virus enters the lytic cycle and upon cytolytic destruction of glial cells, causes the fatal demyelinating disease of the central nervous system (CNS), named progressive multifocal leukoencephalopathy (PML). In this short review, we discuss the molecular pathogenesis of PML by highlighting the role of the immune system in modulating JCV gene activation and replication, and the latency/reactivation of this virus upon immunosuppression. Further, due to the higher incidence of PML among AIDS patients, we further elaborate on the cross-talk between JCV and HIV-1 by direct and indirect pathways that lead to enhanced expression of the JCV genome.     

Identification of critical elements within the JC virus DNA replication origin.

The T antigen of JC virus (JCV) does not interact productively with the simian virus 40 (SV40) origin of replication. In contrast, the SV40 T antigen does drive replication from the JCV origin as well as from its own. The basis for this restricted interaction was investigated by analyzing the structure of the JCV replication origin. The replication activities of JCV-SV40 hybrid origin plasmids were tested in cells constitutively producing either the JCV or SV40 T antigen. Results indicated that a region of the JCV origin critical for interaction with the JCV T antigen was positioned to the late side of the central palindrome of the putative core origin. A mutational analysis of this region indicated that the sequence of the A + T-rich tract was primarily responsible for determining the efficiency with which JCV can initiate replication from its origin. The tandemly repeated pentameric sequence AGGGA located proximal to the A + T-rich tract in the JCV enhancer element was found to stimulate JCV, but not SV40, T antigen-mediated replication. The effect on replication of other elements within the JCV enhancer was also dependent on the T antigen employed for initiation. A plasmid containing the replication origin of prototype BK virus was unable to replicate in cells containing JCV T antigen, again indicating the inflexibility of the JCV T antigen in interacting with heterologous origins.     

Amplification of a bovine papillomavirus-simian virus 40 chimera.

A chimeric plasmid, pBOP, containing bovine papillomavirus (BPV) and the origin of replication from simian virus 40 (SV40) was constructed. The plasmid was established in mouse cells, where it was maintained stably as an autonomous BPV replicon. Lines carrying pBOP were fused to cells of COS-7, a simian line producing SV40 T antigen. Replication dependent on the SV40 origin and having the kinetics and approximate amplitude of an SV40 infection ensued. SV40 replication is therefore dominant over BPV replication, and the SV40 origin can conveniently be used to amplify lower-copy-number plasmids in mammalian cells.     

Requirement for the second coding exon of Tat in the optimal replication of macrophage-tropic HIV-1

HIV-1 Tat is essential for virus replication and is a potent transactivator of viral gene expression. Evidence suggests that Tat also influences virus infectivity and cytopathicity. Here, we find that the second coding exon of Tat contributes a novel function for the replication/infectivity of macrophage-tropic HIV-1. We show that macrophage-tropic HIV-1 which expresses the full-length two-exon form of Tat replicates better in monocyte-derived macrophages (MDM) than an otherwise isogenic virus which expresses only the one-exon form of Tat. Similarly, two-exon Tat expressing HIV-1 also replicates better than one-exon Tat expressing HIV-1 in two different models of human cells/tissue reconstituted SCID mice.     

JC virus enters human glial cells by clathrin-dependent receptor-mediated endocytosis

The human polyomavirus JC virus (JCV) is the etiologic agent of a fatal central nervous system (CNS) demyelinating disease known as progressive multifocal leukoencephalopathy (PML). PML occurs predominantly in immunosuppressed patients and has increased dramatically as a result of the AIDS pandemic. The major target cell of JCV infection and lytic replication in the CNS is the oligodendrocyte. The mechanisms by which JCV initiates and establishes infection of these glial cells are not understood. The initial interaction between JCV and glial cells involves virus binding to N-linked glycoproteins containing terminal alpha(2-6)-linked sialic acids. The subsequent steps of entry and targeting of the viral genome to the nucleus have not been described. In this report, we compare the kinetics and mechanisms of infectious entry of JCV into human glial cells with that of the related polyomavirus, simian virus 40 (SV40). We demonstrate that JCV, unlike SV40, enters glial cells by receptor-mediated clathrin-dependent endocytosis.     

CRISPR/Cas9 System as an Agent for Eliminating Polyomavirus JC Infection

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system (CNS) caused by reactivation of the human polyomavirus JCV gene expression and its replication in oligodendrocytes, the myelin producing cells in the brain. Once a rare disease seen in patients with lymphotproliferative and myeloproliferative disorders, PML has been seen more frequently in HIV-1 positive/AIDS patients as well as patients undergoing immunomodulatory therapy due for autoimmune disorders including multiple sclerosis, rheumatoid arthritis, and others. As of now there is no cure for PML and in most cases disease progression leads to death within two years. Similar to other polyomaviruses, the JCV genome is small circular double stranded DNA that includes coding sequences for the viral early protein, T-antigen, which is critical for directing viral reactivation and lytic infection. Here, we employ a newly developed gene editing strategy, CRISPR/Cas9, to introduce mutations in the viral genome and, by inactivating the gene encoding T-antigen, inhibit viral replication. We first used bioinformatics screening and identified several potential targets within the JCV T-antigen gene that can serve as sites for the creation of guide RNAs (gRNAs) for positioning the Cas9 nuclease on the designated area of the viral genome for editing. Results from a series of integrated genetic and functional studies showed that transient or conditional expression of Cas9 and gRNAs specifically targets the DNA sequences corresponding to the N-terminal region of T-antigen, and by introducing mutation, interferes with expression and function of of the viral protein, hence suppressing viral replication in permissive cells. Results from SURVEYOR assay revealed no off-target effects of the JCV-specific CRISPR/Cas9 editing apparatus. These observations provide the first evidence for the employment of a gene editing strategy as a promising tool for the elimination of the JCV genome and a potential cure for PML.     

人免疫缺陷病毒1型TAT突变蛋白及反义TAT RNA对TAT反式激活作用的抑制

Ｔａｔ是人免疫缺陷病毒（ＨＩＶ）基因组编码的反式激活因子,突变分析表明它含有几个重要的功能域。为寻找控制ＨＩＶ复制的途径,构建了以ＨＩＶ－１ＬＴＲ（－１５８－＋８０）为启动子的Ｔａｔ ｃＤＮＡ全长反义表达质粒ｐＡＳ－Ｔａｔ,并用已经构建的ＨＩＶ ＬＴＲ－１５８到＋８０为启动子,具有不同突变点的突变Ｔａｔ基因表达质粒,以荧光酶基因为报告基因,共转染Ｊｕｒｋａｔ细胞,结果发现无论是反义Ｔａｔ表达质粒还     

Construction and characterization of an SV40 mutant defective in nuclear transport of T antigen

An SV40-adenovirus 7 hybrid virus, PARA(cT), has been described that is defective for the nuclear transport of SV40 large tumor antigen. An SV40(cT) mutant was constructed using SV40 early and late region DNA fragments derived from PARA(cT) and wild-type SV40 respectively. The SV40(cT)-3 construct is defective for viral replication, but can be propagated in COS-1 cells. T antigen induced by SV40(cT)-3 is localized in the cytoplasm of infected cells. The cT mutation also inhibits the transport of wild-type T antigen; COS-1 cells lose their constitutive expression of nuclear T antigen after infection with SV40(cT)-3. Sequence analysis revealed that the cT mutation results in the replacement of a positively charged lysine in wild-type T antigen with a neutral asparagine at amino acid number 128, demonstrating that the alteration of a single amino acid is sufficient to abolish nuclear transport. Implications of the cT mutation on possible mechanisms for the transport of proteins to the nucleus are discussed.