Archetype JC virus efficiently replicates in COS-7 cells, simian cells constitutively expressing simian virus 40 T antigen

Abstract

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.

FIG. 1

Detection of JCV regulatory regions from COS-7 cells transfected with various cloned JCV DNAs. COS-7 cells were transfected with cloned archetype JCV DNAs (CY, MY, C1, SP-1, GH-1, and ZA-1) and a cloned PML-type JCV DNA (Mad-1) and cultured for the indicated number of days. Viral DNAs extracted from the cells were diluted 100 times with water and used as the template to amplify the JCV regulatory region. Amplification was performed with 20 cycles under the conditions described in Materials and Methods. Serially diluted JCV DNA clone CY was used as the control for amplification. The products were resolved by electrophoresis on a 1.5% agarose gel stained with ethidium bromide. The sizes of the HinfI fragments of pUC19 are indicated to the left.

FIG. 2

Restriction analysis of JCV DNAs cloned from COS-7 cells transfected with various archetype JCV DNAs. COS-7 cells were transfected with cloned archetype JCV DNAs (CY, MY, C1, SP-1, GH-1, and ZA-1) and a cloned PML-type JCV DNA (Mad-1) and cultured for 28 days. Viral DNAs extracted from the cells were digested with BamHI, which cleaves the JCV DNA at a single site. The recovered DNAs were cloned into pUC19 as described in Materials and Methods. The recombinant plasmids obtained and the JCV DNAs (Ref) that were used for transfection were digested with a combination of BamHI and HincII. The digests were resolved by electrophoresis on 1.5% agarose gels stained with ethidium bromide. Results with representative clones derived from CY- and MY-transfected COS-7 cells are shown. The sizes of some HinfI fragments of pUC19 and some HindIII fragments of lambda DNA are indicated at the left.

FIG. 3

Immunofluorescence staining of COS-7 cells infected with urinary JCV with rabbit JCAb1 serum and fluorescein-labeled anti-rabbit immunoglobulin. JCAb1 is an antibody against a peptide of JCV capsid protein (VP1) without cross-reaction to BKV (6). Cells infected with JCV from the urinary sediment containing cell-associated JCVs were plated onto a 13-mm-diameter coverslip and processed for immunofluorescence microscopy on day 15 after infection. Densely (D) and sparsely (S) distributed fluorescences are indicated (see text). Magnification, ×200.