Richard J. Frisque

Identification and Characterization of Mefloquine Efficacy against JC Virus In Vitro

ABSTRACT Progressive multifocal leukoencephalopathy (PML) is a rare but frequently fatal disease caused by the uncontrolled replication of JC virus (JCV), a polyomavirus, in the brains of some immunocompromised individuals. Currently, no effective antiviral treatment for this disease has been identified. As a first step in the identification of such therapy, we screened the Spectrum collection of 2,000 approved drugs and biologically active molecules for their anti-JCV activities in an in vitro infection assay. We identified a number of different drugs and compounds that had significant anti-JCV activities at micromolar concentrations and lacked cellular toxicity. Of the compounds with anti-JCV activities, only mefloquine, an antimalarial agent, has been reported to show sufficiently high penetration into the central nervous system such that it would be predicted to achieve efficacious concentrations in the brain. Additional in vitro experiments demonstrated that mefloquine inhibits the viral infection rates of three different JCV isolates, JCV(Mad1), JCV(Mad4), and JCV(M1/SVEΔ), and does so in three different cell types, transformed human glial (SVG-A) cells, primary human fetal glial cells, and primary human astrocytes. Using quantitative PCR to quantify the number of viral copies in cultured cells, we have also shown that mefloquine inhibits viral DNA replication. Finally, we demonstrated that mefloquine does not block viral cell entry; rather, it inhibits viral replication in cells after viral entry. Although no suitable animal model of PML or JCV infection is available for the testing of mefloquine in vivo, our in vitro results, combined with biodistribution data published in the literature, suggest that mefloquine could be an effective therapy for PML.

The Molecular Biology of JC Virus, Causative Agent of Progressive Multifocal Leukoencephalopathy

The rare demyelinating brain disease, progressive multifocal leukoencephalopathy (PML), was first described by Astrom and coworkers (1) in 1958, and a viral etiology was suggested by Zu Rhein and Chou (2) and Silverman and Rubinstein (3) in 1965 based on electron microscopic analysis of infected cells. In 1971, Padgett et al. (4) reported the isolation of a virus from the diseased brain tissue obtained at autopsy from a PML patient. Some have assumed incorrectly that the name given to this virus, JC virus (JCV), identifies it as the agent of Creutzfeldt-Jacob disease, but the name was simply derived from the initials of the PML patient. Structural and antigenic studies (4, 5) demonstrated that JCV was a previously unrecognized member of the genus Polyomavirus within the family Papovaviridae. Additional members of this genus include the closely related human and monkey viruses, BK virus (BKV) and SV40, and the more distantly related type-species, mouse polyomavirus. No evidence has been presented to associate BKV with PML disease. Also, although there have been reports of an SV40 association with human PML (6, 7, 8, 9, 10, 11), we believe there is now sufficient evidence to cast doubt on an SV40 etiology for PML and overwhelming data indicating that JCV is the cause of this fatal disease.

Origin-defective Mutants of SV40

We have described a new technique for the isolation and propagation of nonconditionally lethal mutants. We have used this method to generate mutants of SV40 that contain a defective origin of DNA replication. Using these mutants, we have established the following: (1) SV40 DNA replication and early transcription are functionally separate. (2) A functional viral origin of DNA replication is not necessary for the maintenance of transformation. (3) The lack of the origin of SV40 DNA replication does not affect the efficiency of transformation when nonpermissive cells are transfected by DNA using the calcium technique.

Detection of archetype and rearranged variants of JC virus in multiple tissues from a pediatric PML patient

JC virus (JCV) establishes persistent infections in its human host, and in some immunocompromised individuals, the virus causes the fatal brain disease progressive multifocal leukoencephalopathy (PML). Two forms of the virus, archetype and rearranged, have been isolated, with the latter being derived from the archetype form by deletion and duplication of sequences within the viral transcriptional control region (TCR). We have used the PCR technique to amplify JCV TCR sequences present within multiple tissues of a pediatric PML patient and have cloned and sequenced the PCR products. Archetype JCV was readily detected in kidney tissue; this form of JCV was also identified for the first time in brain and lymph node tissue by employing archetype‐specific PCR primers. In addition, several archetype‐like variants containing small deletions within their regulatory regions were isolated from cardiac muscle and lung. Different, but related rearranged forms were detected in most of the tissues examined. Each of the rearranged TCRs lacked portions of a 66 base pair (bp) region found within the archetype promoter‐enhancer but retained a 23 bp region that is deleted in the prototype (Mad 1) rearranged form of JCV. Although several rearranged forms of JCV were identified in this patient, the TCRs could be assigned to one of two groups based upon the deletion boundaries generated during the adaptation from archetype to rearranged JCV. This study is the first to characterize multiple JCV variants present in different tissues from a patient likely to have succumbed to PML during a primary infection. J. Med. Virol. 52:243–252, 1997.

JC virus T' proteins encoded by alternatively spliced early mRNAs enhance T antigen-mediated viral DNA replication in human cells

Alternative splicing of the JC Virus (JCV) precursor early mRNA yields five transcripts that encode proteins that regulate the life cycle of this human polyomavirus. Large T protein (TAg) mediates viral DNA replication and oncogenic activities, and small t protein influences these functions under certain conditions. Recently, three new early proteins, T′135,T′136, andT′165, were discovered that contain sequences overlapping amino-terminal TAg functional domains. Initial studies with the T′ proteins suggested they contribute to viral DNA replication and transformation. Mutation of a donor splice site utilized by all three T′ mRNAs creates a mutant that exhibits a 10-fold decrease in viral DNA replication compared to wild type JCV. To assess the influence that individual T′ proteins have on the replication process, a set of T′ acceptor site mutants was created in which the unique second acceptor splice site of each T′ mRNA was altered to eliminate production of one, two or all three T′ mRNAs. The patterns of early mRNA and protein expression in these seven mutants were examined, and it was found that mutation of the T′135 acceptor site resulted in the utilization of cryptic splice sites and the generation of new T′ species. Additional mutations were made to prevent these aberrant splicing reactions prior to measuring DNA replication potential of the mutants. DpnI assays revealed that each T′ protein contributes to TAg-mediated DNA replication activity. The three single mutants that express two T′ proteins and the double mutant that only produces T′136, exhibited levels of replication equivalent to that of wild type virus, whereas the two double mutants that fail to express T′136 replicated about twofold less efficiently than wild-type JCV. Replication activity of the triple acceptor site mutant, like that of the T′ donor site mutant from an earlier study, was impaired significantly.

Identification of JC virus variants in multiple tissues of pediatric and adult PML patients

The transcriptional control region (TCR) of JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), undergoes rearrangement during replication of the virus in its human host. The mechanism by which viral promoter/enhancer sequences are deleted and duplicated within the TCR of the archetype form of JCV is not understood, but it is hypothesized that the generation of JCV variants with rearranged TCRs contributes to the viruss pathogenic potential. In a recent study of a pediatric PML patient, we detected extensive rearrangement of the JCV TCR in multiple tissues, and the archetype TCR was amplified from sites other than the kidney. These findings differed from those of previous studies that had examined tissues from adult PML patients. Since exposure to JCV usually occurs early in life, it is likely that some pediatric cases of PML arise as the result of a primary infection, whereas adult cases of PML are thought to result from the reactivation of an infection suffered as an immuno‐ competent child. To investigate further whether rearrangement of the JCV TCR is affected by the hosts age and immune status at the time of exposure, a second pediatric patient and two adult PML patients were examined. As in our first study, multiple tissues were found to contain JCV DNA; however, fewer rearranged variants were detected. In one adult patient, related rearranged variants were detected in the brain, while archetype JCV was found in the other tissues. Based on differences in their VP1 sequences, these two forms represented different JCV genotypes, indicating that this patient had suffered a dual infection. The relevance of these findings to the rearrangement process that alters the JCV TCR is discussed. J. Med. Virol. 58:79–86, 1999.

Identification of SV40 in brain, kidney and urine of healthy and SIV-infected rhesus monkeys

Recent reports of simian virus 40 (SV40) sequences in human tumors have prompted investigations into the poorly understood association of this polyomavirus with its primate host, the rhesus monkey (Macaca mulatta). In the present study we have used PCR to analyze tissues from 20 monkeys for the presence of SV40. Five of the animals, which were infected with simian immunodeficiency virus (SIV), were found to exhibit SV40-induced lesions and to have SV40 sequences present in their kidney and brain. Lesions associated with SV40 were not observed in 15 SIV monkeys, and SV40 DNA was detected in kidney and urine of only one of these animals. Three regions of SV40 DNA were examined in each tissue: the non-coding transcriptional control region (TCR), the sequences encoding the host range domain (HRD) within the carboxy-terminus of T antigen (TAg), and a portion of the VP1 gene. Each region contained nucleotide alterations compared to the SV40 reference strain 776. In all six animals, the TCR had an archetype structure containing a single 72 bp enhancer element. In addition, the TCR amplified from two animals lacked one of three copies of a GC-rich 21 bp repeat which is part of the promoter in strain 776. Multiple clones of unique HRD sequences were derived from different animals, and in some instances from the same animal. No correlation was found between a particular HRD sequence and its presence in a specific tissue type. Nucleotide changes identified within the VP1 gene indicate that this region, as with the closely-related human polyomavirus JCV, may permit the typing of the virus into individual strains. This study is the first to characterize SV40 sequences present in both healthy and SIV-infected animals and supports the suggestion that strain 776 is not the predominant type of SV40 circulating in its natural host.

JC Virus Small t Antigen Binds Phosphatase PP2A and Rb Family Proteins and Is Required for Efficient Viral DNA Replication Activity

Background The human polyomavirus, JC virus (JCV) produces five tumor proteins encoded by transcripts alternatively spliced from one precursor messenger RNA. Significant attention has been given to replication and transforming activities of JCVs large tumor antigen (TAg) and three T′ proteins, but little is known about small tumor antigen (tAg) functions. Amino-terminal sequences of tAg overlap with those of the other tumor proteins, but the carboxy half of tAg is unique. These latter sequences are the least conserved among the early coding regions of primate polyomaviruses. Methodology and Findings We investigated the ability of wild type and mutant forms of JCV tAg to interact with cellular proteins involved in regulating cell proliferation and survival. The JCV P99A tAg is mutated at a conserved proline, which in the SV40 tAg is required for efficient interaction with protein phosphatase 2A (PP2A), and the C157A mutant tAg is altered at one of two newly recognized LxCxE motifs. Relative to wild type and C157A tAgs, P99A tAg interacts inefficiently with PP2A in vivo. Unlike SV40 tAg, JCV tAg binds to the Rb family of tumor suppressor proteins. Viral DNAs expressing mutant t proteins replicated less efficiently than did the intact JCV genome. A JCV construct incapable of expressing tAg was replication-incompetent, a defect not complemented in trans using a tAg-expressing vector. Conclusions JCV tAg possesses unique properties among the polyomavirus small t proteins. It contributes significantly to viral DNA replication in vivo; a tAg null mutant failed to display detectable DNA replication activity, and a tAg substitution mutant, reduced in PP2A binding, was replication-defective. Our observation that JCV tAg binds Rb proteins, indicates all five JCV tumor proteins have the potential to influence cell cycle progression in infected and transformed cells. It remains unclear how these proteins coordinate their unique and overlapping functions.

JC virus T'135, T'136 and T'165 proteins interact with cellular p107 and p130 in vivo and influence viral transformation potential.

The JC virus (JCV) regulatory proteins, large T antigen, small t antigen, T′135, T′136, and T′165, are encoded by five transcripts alternatively spliced from the viral early precursor mRNA. T antigen and the T′ proteins share N-terminal amino acid sequences that include the L × C × E and J domains, motifs in SV40 T antigen known to mediate binding to the retinoblastoma (Rb) proteins and Hsc70, respectively. In this study, G418-resistant cell lines were created that express wild-type or mutant JCV T antigen and T′ proteins individually or in combination. These cell lines were used to evaluate the ability of each viral protein to bind p107 and p130 in vivo, and to influence cellular growth characteristics. Differences were observed in the abilities of individual T′ proteins to bind p107 and p130 and to alter their phosphorylation status. The T′ proteins were also found to localize to the cell’s nucleus and to be phosphorylated in a cell cycle-dependent manner. JCV T antigen and T′ proteins expressed from a cytomegalovirus promoter failed to induce dense focus formation in Rat2 cells, but they did cooperate with a mutant Ras protein to overcome cellular senescence and immortalize rat embryo fibroblasts. These data indicate that, despite their sequence similarities, JCV early proteins exhibit unique activities that, in combination, effect the inactivation of cell cycle regulators, a requirement for polyomavirus-induced transformation.

Altered DNA binding and replication activities of JC Virus T-antigen mutants

Ten mutations were introduced into the JC virus (JCV) T antigen within a region corresponding to the SV40 T-antigen DNA binding domain (SV40 amino acids 131 to 220); nine of these increased homology between the two proteins in sequences critical for SV40 T antigen DNA binding. All mutant JCV T antigens bound to JCV and SV40 origins of DNA replication. Binding efficiency relative to the of wild-type JCV T antigen ranged from 83 to 301% for the JCV binding sites and from 44 to 240% for the SV40 binding sites. Nine mutant proteins promoted viral DNA replication in primary human fetal glial (PHFG) and CV-1 cells. In PHFG cells, promotion of DNA replication ranged from 26 to 220% relative to that of wild-type T antigen; in CV-1 cells it ranged from 14 to 522%. Coding sequences for five mutant proteins were transferred into the hybrid virus M1 (SV40) [M1(SV40) contains coding sequences from JCV and regulatory sequences from SV40]. Wild-type T antigen promoted replication weakly from the SV40 origin in these hybrid viruses in CV-1 cells (2% that from the JCV origin); replication driven by the mutant proteins ranged from 110 to 412% of that induced by the wild-type protein. Efficient specific DNA binding by a mutant T antigen was not a reliable indicator of that mutant proteins ability to promote DNA replication.