Sequences within the Early and Late Promoters of Archetype JC Virus Restrict Viral DNA Replication and Infectivity

VIROLOGY

216, 90–101 (1996) 0037

ARTICLE NO.

Sequences within the Early and Late Promoters of Archetype JC Virus Restrict Viral DNA Replication and Infectivity ANN MARIE DANIEL,* JENNIFER J. SWENSON,* RAVI P. REDDY MAYREDDY,† KAMEL KHALILI,† and RICHARD J. FRISQUE*,1 *Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, 16802; and †Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 Received October 5, 1995; accepted November 27, 1995 Two forms of JC virus (JCV) have been isolated from its human host, an archetype found in kidney tissue and urine of nonimmunocompromised individuals and a rearranged type detected in lymphocytes and brain tissue of patients with and without progressive multifocal leukoencephalopathy. To investigate the hypothesis that alterations to the archetype transcriptional control region yield rearranged forms of the virus exhibiting new tissue tropic and pathogenic potentials, attempts were made to propagate archetype JCV in human renal and glial cell cultures. Although rearranged forms of JCV multiplied in these cells, archetype JCV failed to do so. Through the use of chimeric and mutant viral genomes, and a cell line that constitutively expresses viral T protein, we demonstrated that archetype’s inactivity relative to that of rearranged forms was due to differences in the promoter–enhancer and not in the protein coding regions or origin of DNA replication. Additional analyses revealed that the absence of a large tandem duplication and the presence of a 23- and a 66-base pair sequence in the archetype transcriptional control region were responsible for this restricted lytic behavior. We discuss the possibility that deletion and duplication events within the archetype promoter–enhancer might yield more active viral variants via the loss of a negative, or the creation of a positive, transcriptional control signal(s). q 1996 Academic Press, Inc.

INTRODUCTION

urine (Hogan et al., 1980); recently JCV has also been detected in the brain of normal individuals (Elsner and Do¨rries, 1992; Mori et al., 1992; Quinlivan et al., 1992; White et al., 1992). In severely immunodeficient patients, a subclinical infection might be reactivated, leading to the fatal demyelinating brain disease progressive multifocal leukoencephalopathy (PML). Reactivation may result under conditions that affect T-cell-mediated immune surveillance (Walker and Frisque, 1986) or the production of cellular factors, such as cytokines, which influence viral gene expression (Ranganathan and Khalili, 1993; Raj and Khalili, 1994; Atwood et al., 1995). Significantly, such conditions occur in AIDS patients and approximately 4% of these individuals succumb to PML (Krupp et al., 1985; Berger et al., 1987). Two types of JCV have been found in human tissues. Archetype JCV is detected in the kidneys and urine of people with and without PML (Loeber and Do¨rries, 1988; Yogo et al., 1990); it is thought to be the type of JCV that circulates in the population. Little sequence variation is observed in the genomes of independent isolates of this form of the virus (Yogo et al., 1990, 1991). It has been suggested that during viral replication, sequence rearrangements occur within the archetype transcriptional control region (TCR), yielding a new, potentially more active form of the virus. Multiple examples of this rearranged type of JCV have been isolated from the brain and lymphocytes of people with and without PML (Torna-

Serological data indicate that about half of the world’s population is infected by JC virus (JCV) during childhood and that eventually 90% of the population is exposed to the virus (Frisque and White, 1992; Major et al., 1992). The route of transmission has not been established, due in part to the asymptomatic nature of the primary infection. Evidence for a respiratory (Sundsfjord et al., 1994) or transplacental (Mori et al., 1992; Do¨rries et al., 1994; Kitamura et al., 1994) route is lacking; however, viruria is a common event, especially in immunocompromised individuals (Frisque and White, 1992; Tornatore et al., 1994), and transmission via a urine to oral route has been proposed (Walker and Frisque, 1986). The primary site of infection in the body also remains unknown. Recent PCR results suggest that JCV does not enter through cells lining the upper respiratory or alimentary tracts (Sundsfjord et al., 1994), and the authors suggest that the lower alimentary tract should be considered a portal of entry. Most of what is known about the interaction of JCV with its human host involves events occurring subsequent to the initial exposure. We have known for a number of years that in healthy individuals JCV reaches the kidney, establishes a persistent infection, and is excreted in the 1 To whom correspondence and reprint requests should be addressed.

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Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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tore et al., 1992; White et al., 1992; Do¨rries et al., 1994). The TCR of the Mad1 strain of JCV, the first rearranged form isolated from PML brain tissue (Padgett et al., 1971), includes a 98-base pair (bp) tandem repeat, with each copy containing multiple binding sites for factors that affect basal and activated levels of transcription (for review see Raj and Khalili, 1995). The TCRs of other strains of rearranged JCV obtained from PML tissues have been shown to exhibit considerable variation in the arrangement of these transcription signals (Martin et al., 1985). A comparison of the TCRs of archetype and each of the rearranged forms suggests that the latter forms were generated by deletion and duplication of the archetype promoter–enhancer sequences (Ault and Stoner, 1993; Iida et al., 1993). In the case of Mad1, the TCR differs from that of archetype by its duplicated structure and its lack of two blocks of DNA, a 23- and a 66-bp sequence. Biological studies of the JCV polyomavirus have focused upon the prototype Mad1 strain. Mad1 demonstrates a highly restricted lytic behavior in cell culture, replicating well only in primary human fetal glial (PHFG) cells and their transformed derivatives (Padgett et al., 1971; Mandl et al., 1987). Lytic function and tissue tropism are regulated in part at the level of transcription initiation (Kenney et al., 1984; Feigenbaum et al., 1987). The underlying question that has stimulated our interest in archetype JCV asks: Does archetype represent a persistent, less active form of JCV which, upon rearrangement, acquires a new tissue tropism and pathogenic potential? To date, the biological activity of archetype JCV has not been characterized in vitro. In this report we examine its replication activity in human fetal glial and adult kidney proximal tubule cells. To identify portions of the archetype genome that contribute to its unique behavior, we have utilized mutant and chimeric viruses as well as two naturally occurring JCV variants, Mad8Br and Mad11Br. The TCRs of these latter two rearranged forms possess structures which are intermediate between those of Mad1 and archetype. MATERIALS AND METHODS Cells and media Adult human kidney proximal tubule (HPT) cells, and culture medium and protocols for their propagation, were kindly provided by Drs. John Todd and Donald Sens (West Virginia University Health Science Center; Todd et al., 1995). HPT cells were grown on collagen-treated culture flasks or plates in a 1:1 mixture of Dulbecco modified Eagle medium (DMEM) and Ham F-12 medium containing insulin (5 mg/ml), transferrin (5 mg/ml), selenium (5 ng/ml), hydrocortisone (36 ng/ml), triiodothyronine (4 pg/ml), penicillin (100 U/ml), streptomycin (10 mg/ml), fungizone (250 ng/ml), human recombinant epidermal growth factor (10 ng/ml), and L-glutamine (292 mg/ml) at 377 in a humidified atmosphere containing 5% CO2 . PHFG

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cells were processed as described previously (Padgett et al., 1977) and were maintained in DMEM containing penicillin (99 U/ml), streptomycin (73 U/ml), and fungizone (2.5 mg/ml) and supplemented with 3% bovine calf serum at 377 in a humidified atmosphere containing 10% CO2 . POJ-19 cells (PHFG cells transformed by an origindefective mutant of JCV; Mandl et al., 1987) and U-87MG cells (a continuous line of human glioblastoma cells obtained from ATCC) were grown in DMEM containing penicillin (99 U/ml) and streptomycin (73 U/ml) and supplemented with 10% fetal bovine serum (FBS). Viral recombinant DNAs The Mad1 prototype strain of JCV, JCV(Mad1), was isolated from PML brain tissue (Padgett et al., 1971), and its DNA cloned into the unique EcoRI site of pBR322 (pMad1TC; Frisque, 1983). The CY strain of archetype JCV, JCV(CY), was isolated from the urine of a healthy individual, and its DNA cloned into the BamHI site of pUC-19 (pJC-CY; kindly provided by Dr. Yoshiaki Yogo, The University of Tokyo; Yogo et al., 1990). JCV(Mad1) and JCV(CY) will be referred to as Mad1 and CY, respectively, in this article. The recombinant plasmids pM10 and pCY0 were constructed by inserting an NcoI restriction fragment encompassing the TCR (includes viral replication origin and promoter–enhancer) of Mad1 and CY, respectively, into the vector pKP55 (Li and Kelly, 1985; Lynch and Frisque, 1990). Production of pM1D98 and pM1D980 entailed the digestion of pMad1TC and pM10 with SacI, respectively, to remove one of the 98-bp tandem repeats from the Mad1 TCR (Bollag et al., 1989; Lynch and Frisque, 1990). Two chimeric viral TCRs, pCYD230 and pCYD660 , were created by digesting pM1D980 and pCY0 with ClaI/SacI and exchanging the resulting 218- and 245-bp fragments. These recombinant DNAs lack either the 23-bp (pCYD230) or the 66-bp (pCYD660) sequences found in the TCR of CY but not of Mad1. NcoI fragments containing the TCRs of pCY0 , pCYD230 , and pCYD660 were inserted into the unique NcoI site of pM1DNcoI, a clone of Mad1 lacking the entire TCR (Bollag et al., 1989), to generate pM1(CY), pM1(CYD23), and pM1(CYD66), respectively. These three chimeric plasmids contain full-length viral genomes in which the archetyperelated TCRs (in parentheses) are linked to the Mad1 coding region. To construct the recombinant chimeric genome, pCY(M1), a 416-bp NcoI fragment from pJC-CY was replaced with the corresponding 425-bp fragment of pMad1TC. Recombinant DNAs pMad8Br and pMad11Br were produced by cloning viral DNA directly from the brain tissues of two PML patients into the EcoRI site of pBR322 (Grinnell et al., 1983). Exchanging the NcoI restriction fragments among pMad1-TC, pMad8Br, and pMad11Br generated the JCV chimeras pM1(M8), pM8(M1), pM1(M11), and pM11(M1); the viral TCR is enclosed within the parentheses. The TCRs of the JCV genomes analyzed in this study are depicted in Fig. 1.

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FIG. 1. Structural arrangements of various JCV TCRs relative to that of the archetype CY strain of JCV isolated from the urine of a healthy individual (Yogo et al., 1990). Mad1, Mad4, Mad11Br, and Mad8Br are naturally occurring rearranged forms of JCV isolated from PML brain tissue (Padgett et al., 1971; Grinnell et al., 1983). CYD66, CYD23, and M1D98 were laboratory constructs. Vertical numbers above the diagram indicate the nucleotide position according to the numbering system described in Frisque et al. (1984), and horizontal numbers indicate number of nucleotides in defined regions of the archetype TCR. The TATA box is contained within the 25-bp region. The two NcoI (N) restriction sites were used in the present study to exchange viral TCRs between JCV variants; the SacI (S) site was used to exchange CY and M1D98 sequences to create the chimeric TCRs in M1(CYD66) and M1(CYD23). The NcoI site at nucleotide position 275 contains the initiation codon for the late agnoprotein. Sequences in the various TCRs that are identical to those in CY are represented by solid horizontal lines, deleted sequences are indicated by gaps between lines, and duplicated sequences are denoted as parallel lines. In the case of repeated elements, the reading of a sequence begins at the left and continues all the way to the right before starting to the left of the second line.

To create JCV–CAT constructs, viral TCR sequences were amplified by PCR using Primers 1, 2, and 3, representing JCV nucleotides 226–253, 4982–5010, and 4989–5010, respectively. Sequences amplified by Primers 1 and 2 (Table 3) or Primers 1 and 3 (Table 5) were cloned into the BamHI site of the promoterless reporter plasmid pBLCAT3 (Luckow and Schutz, 1987). DNA transfections and viral infections Cultures of HPT, POJ-19, and PHFG cells were 80 to 100% confluent at the time of transfection. Viral DNA was released from the pBR322 and pUC-19 vectors using EcoRI and BamHI, respectively, and recircularized via ligation prior to the transfection of cells. Cells grown on 60-mm plates were transfected with 125 ng of the intact viral genomic DNAs, recombinant pKP55 plasmids containing the various TCRs, or calf thymus DNA using a modified DEAE–dextran procedure (Sompayrac and Danna, 1981). Following a 2-hr incubation at 377, the transfected HPT, POJ-19, and PHFG cells were washed with DMEM, and the appropriate medium was added, changed the following day, and then replaced every 3,

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5, and 7 days, respectively. To test for the production of infectious virus in these cells, 0.1 ml of a cellular extract (4.0 ml total volume) obtained from Day 21 posttransfected cultures was used to infect a fresh culture of cells (Myers et al., 1989). DNA replication assay DNA replication in JCV-transfected and infected cells was examined using a modified DpnI assay (Peden et al., 1980). At various times posttransfection (p.t.) or postinfection (p.i.), low-molecular-weight DNA was extracted from the cells according to the Hirt procedure (Hirt, 1967). DNA was digested with the restriction enzymes DpnI plus EcoRI or BamHI, and resulting fragments were separated by electrophoresis on a 0.8% agarose gel, transferred onto GeneScreen Plus nylon membrane (DuPont), and hybridized at 657 in a hybridization incubator (Robbins Scientific) to 32P-labeled (Pharmacia oligolabeling kit) pKP55 or pMad1-TC DNA. Linearized pKP55 (1964 bp) or EcoRI/BglI-digested pMad1-TC (5130, 2319, 934, 876 bp) were used as size markers on the gels. Repli-

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cated DNA was detected by autoradiography and analyzed on a betaScope 603 blot analyzer (Betagen).

93 TABLE 1

DNA Replication and Viability of Archetype, Prototype, and Chimeric JCV in HPT Cells

CAT assays Approximately 1 1 106 U-87MG cells were plated onto 60-mm dishes in DMEM plus 10% FBS. Sixteen hours later, CAT vector DNA (5 mg) was mixed with pBR322 plasmid DNA (10 mg), coprecipitated with calcium phosphate in a volume of 1 ml, and added to the cells (Graham and van der Eb, 1973). Cells were incubated with the calcium phosphate–DNA mixture for 4 hr, washed with phosphate-buffered saline, treated with 10% glycerol for 2 min, and then cultured for 36 hr. CAT assays were performed as described previously (Gorman et al., 1982), except that samples of cell extracts were diluted in 0.25 M Tris (pH 7.8) in a final volume of 150 ml. RESULTS JCV DNA replication and infectious virion production in human kidney and glial cells Although archetype JCV DNA is frequently detected in the urine and kidneys of healthy individuals (Yogo et al., 1990; Markowitz et al., 1991), propagation of the virus in cell culture has not been reported. To initiate the present study we sought to identify a permissive cell system in which to investigate the lytic behavior of archetype JCV. Such a finding would allow us to test in vitro the hypothesis that rearrangements to the archetype TCR give rise to the types of JCV found in brain tissue of PML patients. Because of archetype’s association with renal tissue in vivo, we first tested the ability of HPT cells to support viral replication. The cells were transfected with viral DNAs representing archetype JCV (CY strain from normal kidney), rearranged

FIG. 2. DNA replication of CY, CY(M1), and Mad1 in HPT cells. HPT cells were transfected with 125 ng/ml of calf thymus, CY, CY(M1), or Mad1 DNA. Low-molecular-weight DNA was extracted from cells at 0, 7, 14, and 21 days p.t. and digested with DpnI and either EcoRI or BamHI. Digested DNA fragments were separated on a 0.8% agarose gel, transferred onto GeneScreen Plus nylon membranes, hybridized to linearized 32P-labeled pMad1-TC DNA, and exposed to film for 4 days. Positions of bands representing replicated (DpnI-resistant) and input (DpnI-sensitive) DNA are shown.

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Experimenta DNA CY

CY(M1)

Mad1

Day p.t.

1

2

7 14 21 7 14 21 7 14 21

— — — 0.1 1.4 4.8 1.0 31.4 59.4

— — — — 2.3 6.3 1.0 25.6 66.8

Viabilityb

0

{

/

a Numerical values for DNA replication were obtained by betascope analysis of Southern blots of DpnI-digested DNAs and normalizing to Mad1 Day 7 p.t. values (arbitrarily set at 1.0) after background subtraction. (—), no replicated DNA detected in the transfected cells. b Viability was assessed by determining whether replicating DNA appeared in fresh cultures of cells infected with extracts of Day 21 posttransfected cells. (0), no DNA bands detected; ({), faint bands detected in one or both experiments at Days 14 and/or 21 p.i.; (/), bands detected and their intensity increased during the course of the experiment.

JCV (Mad1 strain from PML brain), and CY(M1), a chimera containing archetype coding and Mad1 regulatory sequences. At Days 0, 7, 14, and 21 p.t., cells were examined for the presence of replicating viral DNA (Fig. 2, Table 1). Mad1 replicated its DNA to low levels in these cells; however, CY failed to do so. Although less efficient than Mad1, the chimeric DNA did replicate, suggesting that the archetype proteins were functional when expressed by the Mad1 TCR. Extracts from transfected cells were also added to fresh HPT cell cultures to determine whether infectious virions had been produced; low virus yields were detected only in extracts of cells that had originally received Mad1 or CY(M1) DNA (Table 1). Our next set of experiments tested archetype activity in PHFG cells which are known to be permissive for the growth of rearranged forms of JCV (Padgett et al., 1971). DNA replication and viability experiments were repeated in these cells using four DNAs: CY, Mad1, CY(M1), and M1D98. In PHFG cells, CY exhibited barely detectable DNA replication activity and failed to produce infectious virus (Table 2). On the other hand, Mad1 and CY(M1) efficiently replicated their genomes and generated viable virus. The deletion of one copy of the promoter–enhancer element in M1D98 resulted in reduced DNA replication and virion production relative to that of the parental genome. The above findings were supported by immunofluorescence analysis results in which very few cells transfected with M1D98 or CY contained the JCV replication protein T antigen (TAg) compared with those cells exposed to Mad1 or CY(M1) (data not shown).

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DANIEL ET AL. TABLE 2 DNA Replication and Viability of Archetype, Prototype, Mutant, and Chimeric JCV in PHFG Cells Experimenta

DNA CY

M1D98

CY(M1)

Mad1

Day p.t. 7 14 21 7 14 21 7 14 21 7 14 21

1

2

— õ0.1 õ0.1 1.0 3.9 12.8 7.0 15.4 113.8 8.6 48.9 157.1

— 0.1 — 1.0 11.0 17.0 19.6 71.9 239.5 NAc 168.4 1079.6

Viabilityb

0

{

/

/

a Numerical values for DNA replication were obtained by betascope analysis of Southern blots of DpnI-digested DNAs and normalizing to M1D98 Day 7 p.t. values (arbitrarily set at 1.0) after background subtraction. (—), no replicated DNA detected in the transfected cells. At Days 14 and 21 p.t., Mad1 replicated its DNA in PHFG cells approximately 200 to 400 times more than in HPT cells. These values were determined by comparing replication activities in PHFG (Tables 2 and 4) and HPT cells (Table 1) after normalizing values to the Mad1 size marker band in each experiment (data not shown). b Viability was assessed as described for Table 1. c NA, not available.

JCV DNA replication in POJ cells The nominal replication activity of archetype JCV DNA compared to that of Mad1 suggested a defect in at least one of the two viral components required for replication: the viral origin or the viral signals that specify transcription of the early mRNA encoding the JCV TAg. To test the first of these possibilities, recombinant DNAs were constructed in which the CY and Mad1 origin sequences were cloned into a plasmid vector (pCY0 and pM10 , respectively). Because the major differences between the TCRs of the archetype and rearranged forms of JCV are the presence or absence of (i) a 23- and a 66-bp block of DNA and (ii) a large tandem duplication of the promoter – enhancer sequences (Fig. 1), we created three additional origin plasmids for inclusion in the analysis. The recombinant plasmid pM1D980 contains the M1D98 TCR, and the pCYD230 and pCYD660 constructs contain the archetype TCR minus either the unique 23-bp or the unique 66-bp sequence, respectively (Fig. 1). The five origin plasmids were transfected into POJ-19 cells which constitutively express a functional Mad1 TAg. All of the constructs replicated with similar efficiency (Fig. 3), indicating that the archetype origin sequences were functional and capable of interacting productively with the endogenous TAg. Furthermore, these results suggested that the failure of the intact archetype genome

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to replicate efficiently in HPT and PHFG cells was likely due to the inability of its early promoter to direct the expression of adequate levels of TAg mRNA. We next transfected intact CY and Mad1 genomes into the POJ-19 cell line to determine whether infectious virions would be produced in these cells. Since POJ-19 cells supply TAg in trans, this experiment would not require a functional early promoter of either virus. Instead, it would permit us to determine whether the late promoter of each virus was active. As expected, given the results from the previous experiment with the origin plasmids, both CY and Mad1 replicated their DNAs to similar levels at Day 7 p.t. (Fig. 4A). However, while Mad1 DNA continued to accumulate at the later time points, the level of replicated archetype DNA did not increase. This data suggested that Mad1, but not CY, efficiently produced virus particles capable of causing secondary infection in the cell cultures. This suggestion was confirmed by adding extracts of the CY- and Mad1-transfected cells onto fresh cultures of POJ-19 and PHFG cells and looking for the presence of replicating viral DNA (Figs. 4B and 4C, respectively). As predicted, replicating Mad1 DNA was observed in both types of cells. In addition, the results revealed that some archetype virus had been produced in the original transfection, since CY DNA was detected in both PHFG and POJ-19 cells at Day 0 p.i. However, evidence of a productive archetype infection in PHFG cells was lacking, since DNA replication did not continue into the later time points. On the other hand, low levels of archetype DNA replication were detected in the POJ-19 cells at each time point, indicating that in the presence of endogenous TAg, a small amount of infectious virus continued to be generated. These results suggest that CY viability is limited not only by a relatively inactive early promoter, but also by a defect in late transcription or virion assembly.

FIG. 3. DNA replication of JCV origin plasmids in POJ-19 cells. POJ-19 cells were transfected with 125 ng/ml of pKP55 vector, pCY0 , pCYD660 , pCYD230 , pM1D980 , or pM10 DNA. Low-molecular-weight DNA was extracted from cells at 0, 3, and 7 days p.t. and digested with DpnI and ClaI. The digested DNA fragments were separated on a 0.8% agarose gel, transferred onto GeneScreen Plus nylon membranes, hybridized to linearized 32P-labeled pKP55, and exposed to film for 4 hr. Positions of bands representing replicated (DpnI-resistant) and input (DpnI-sensitive) DNA are shown.

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95

rearranged forms of JCV TCRs, we tested promoter function using the CAT assay (Gorman et al., 1982). CAT vectors containing the TCRs from CY, Mad4 [similar in structure (Fig. 1) and activity (Frisque and White, 1992) to the Mad1 TCR], or M4D79 (identical in structure to M1D98) in either the early or the late orientation were transfected into U-87MG glioblastoma cells. Determination of the levels of CAT activity in the extracts of transfected U-87MG cells indicated that both CY promoters were much less active than those of Mad4 (Table 3). Furthermore, deletion of one copy of the Mad4 promoter – enhancer also resulted in a substantial reduction in CAT activity (compare M4D79-CAT vs Mad4-CAT, Table 3). DNA replication and viability of chimeric CY and Mad1 genomes in PHFG cells The experiments outlined above have revealed that the archetype proteins and replication origin are functional, but that the early and late promoters are inefficient relative to those of Mad1. We then asked which sequences within the TCR were responsible for this inactivity. This region of the archetype genome is thought to undergo deletion and duplication events to yield the rearranged forms of JCV. The archetype TCR is unique in that (i) it is nearly identical in every urine isolate examined from nonimmunosuppressed individuals and (ii) it contains no long stretches of repeated sequences (Fig. 1). The Mad1 TCR, relative to that of CY, has lost the 23- and 66-bp blocks of DNA and then duplicated the remaining 98 bp present in archetype. Most other examples of rearranged forms of JCV TCRs retain the 23-bp block, but lose at least part of the 66-bp block. Interestingly, the 5* end of the 66-bp stretch of DNA has homology to a negative regulatory sequence in the TCR of a second human polyomavirus, BK virus (BKV; Grinnell et

TABLE 3 Transcriptional Activities of Early and Late Promoters of Archetype and Rearranged JCV FIG. 4. DNA replication of CY and Mad1 genomes in POJ-19 and their viability in POJ-19 and PHFG cells. (A) POJ-19 cells were transfected with 125 ng/ml of calf thymus, CY, and Mad1 DNA and examined for the presence of replicating DNA by the DNA replication assay. Extracts of POJ-19 cells (Day 21 p.t.) were used to infect fresh cultures of POJ-19 (B) and PHFG (C) cells; the detection of replicated DNA in these cells at Days 7, 14, and 21 p.i. confirmed that infectious virions were present in the extracts. A description of the DNA replication assay and the identity of the bands is given in the legend to Fig. 2. Films were exposed for 3 (A), 12 (B), or 72 (C) hr.

CAT expression directed by JCV early and late promoters To investigate the possibility that both promoters of the archetype JCV TCR were less active than those of

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DNAa

% Conversionb

Mad4E –CAT M4D79E –CAT CYE –CAT Mad4L –CAT M4D79L –CAT CYL –CAT

13.7 1.6 2.9 11.9 3.8 1.2

a The TCRs of Mad4 [similar in structure (Fig. 1) and activity (Frisque and White, 1992) to Mad1], M4D79 (identical to M1D98), and CY were cloned into pBLCAT3 in both the early (E) and the late (L) orientations. b CAT assays were performed using extracts of U-87MG cells transfected with the various CAT vectors. The value determined for percentage conversion of chloramphenicol into its acetylated forms represents the average of two experiments.

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DANIEL ET AL. TABLE 4 DNA Replication and Viability of JCV Genomes Containing Chimeric TCRs in PHFG Cells Experimenta DNA

Day p.t.

CY

7 14 21 7 14 21 7 14 21 7 14 21 7 14 21 7 14 21 7 14 21

M1(CY) FIG. 5. DNA replication of archetype, prototype, mutant, and chimeric JCV genomes in PHFG cells. PHFG cells were transfected with 125 ng/ ml of calf thymus, CY, CY(M1), M1D98, M1(CY), M1(CYD66), M1(CYD23), and Mad1 DNA. A description of the DNA replication assay and the identity of the bands are given in the legend to Fig. 2. The film was exposed for 12 hr using an intensifier screen.

al., 1988). Based on this information, we predicted that the answer to archetype’s reduced activity would be found in the 66-bp block and the lack of a duplication in its TCR. To test this hypothesis, intact chimeric genomes were constructed in which the Mad1 TCR was replaced with the TCRs from the origin plasmids pCY0 , pCYD230 , and pCYD660 to yield M1(CY), M1(CYD23), and M1(CYD66), respectively. The ability of each DNA to replicate and produce infectious virions in PHFG cells was compared to that of the previously tested (Table 2) CY, M1D98, CY(M1), and Mad1 DNAs (Fig. 5, Table 4). This collection of DNAs allowed us to investigate the influence of the 23- and 66-bp archetype sequences individually. The chimeric M1(CY) DNA, like CY DNA, exhibited minimal activity in PHFG cells. The deletion of the 66-bp region of the CY TCR enhanced the replication efficiency of the M1(CYD66) DNA relative to that of archetype JCV DNA, but it did not result in the appearance of viable virus. With the removal of the 23-bp region, replication of M1(CYD23) DNA was increased over that of M1(CYD66) DNA. This finding is in agreement with a second series of CAT assays which demonstrated that both early and late CYD23 promoters induced higher levels of CAT expression than the corresponding CYD66 promoters (Table 5). Accumulation of replicated M1(CYD23) DNA at the later time points of the DpnI assay also suggested that infectious virions were being generated. Passage of extracts from cells transfected with this DNA indicated that low levels of virus were indeed produced. As expected, M1D98 was viable, but less active than Mad1, confirming our earlier CAT (Frisque and White, 1992) and replication (Fig. 2) data that indicated a single copy of the promoter–enhancer was less efficient than two copies. These results have redirected our attention to the 23-bp block in regards to the restricted lytic behavior of archetype JCV. It should be noted, however, that the lack of a tandem duplication (compare Mad1 vs M1D98, Table 4) and the presence of the 66-bp block [compare M1D98 vs M1(CYD23), Table 4] also contribute to this behavior.

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M1(CYD66)

M1(CYD23)

M1D98

CY(M1)

Mad1

1 õ0.1 õ0.1 õ0.1 õ0.1 õ0.1 õ0.1 0.1 0.9 0.4 0.5 1.4 3.7 1.0 2.5 12.2 5.2 18.1 137.1 9.9 59.3 349.7

2 õ0.1 0.1 õ0.1 õ0.1 õ0.1 õ0.1 õ0.1 0.4 0.5 0.2 0.7 1.6 1.0 2.5 7.3 4.7 7.9 66.2 2.8 10.8 32.4

Viability b

0

0c

0

{

{

/

/

a Numerical values for DNA replication were obtained as described for Table 2. b Viability was assessed as described for Table 1. c After a long exposure, a very faint band was observed in one experiment at Day 21 p.i.

DNA replication and viability of naturally occurring rearranged types of JCV in PHFG cells Our next step in analyzing the archetype TCR was to look at two rearranged forms of JCV that had been cloned directly from PML brain tissue and which differ in their arrangement of the 23- and 66-bp blocks. The Mad8Br and Mad11Br TCRs contain 83- and 40-bp tandem duplications, respectively (Fig. 1). The Mad8Br sequence includes approximately 1.5 copies of the 23-bp region and

TABLE 5 Transcriptional Activities of Chimeric JCV Early and Late Promoters DNA a

% Conversion b

CYE –CAT CYD23E –CAT CYD66E –CAT CYL –CAT CYD23L –CAT CYD66L –CAT

43.2 8.3 3.6 57.6 6.1 0.9

a The CY and chimeric CYD23 and CYD66 TCRs were cloned into pBLCAT3 in both the early (E) and the late (L) orientations. b CAT assays were performed as described in Table 3.

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FIG. 6. DNA replication of prototype, variant, and chimeric JCV genomes in PHFG cells. PHFG cells were transfected with 125 ng/ml of calf thymus, Mad8Br, M1(M8), M8(M1), Mad11Br, M1(M11), M11(M1), and Mad1. A description of the DNA replication assay and identity of the bands is given in the legend to Fig. 2. Film was exposed 14.5 hr.

lacks the 66-bp region, while the Mad11Br sequence includes 1 copy of the 23-bp block and the 3* portion of the 66-bp block (sequences homologous to the BKV negative regulatory element are missing). Although propagation of both variants in PHFG cells has been reported (Grinnell et al., 1983), we have not been able to reproduce this finding (Myers et al., 1989, unpublished results). We have used these two DNAs to make an additional set of four chimeras in which the TCR sequences have been swapped with those of Mad1: M1(M8), M8(M1), M1(M11), and M11(M1). The four chimeric and three parental JCV DNAs were once again tested for the ability to replicate their DNA and to produce infectious virions in PHFG cells (Fig. 6, Table 6). Mad8Br and Mad11Br both replicated their DNAs inefficiently; similar activities were observed for M1(M8) and M1(M11), which were composed of the Mad1 coding region and the Mad8Br or Mad11Br TCRs, respectively. These four genomes yielded little if any infectious virus. The M8(M1) chimeric DNA replicated slightly better than the Mad8Br and M1(M8) DNAs, but it too was greatly limited in its ability to produce viable virus. M11(M1), like Mad1, exhibited efficient DNA replication and was viable.

1992). It has been hypothesized that archetype virus represents the form which circulates in the host population and rearranges to generate the multiple genotypes observed during replication in the infected host or in cultured cells. Further, it is suspected that rearranged forms acquire a new tissue tropism and greater pathogenic potential (Rubinstein et al., 1991; Frisque and White, 1992). In the present study we initiated an investigation of the CY archetype strain of JCV. JCV is the most difficult of the three primate polyomaviruses to propagate in cell culture and while prototype Mad1 grows well only in human fetal glial cells (Padgett et al., 1977), archetype JCV had not been reported to grow in any cultured cells. Given that (i) rearranged forms of JCV replicate to a limited extent in human kidney cells (Miyamura et al., 1985), (ii) polyomaviruses have been identified in renal tubule epithelium in vivo (Do¨rries and ter Meulen, 1983; Greenlee et al., 1991; Ilyinskii et al., 1992; Atencio et al., 1993), and (iii) archetype JCV is detected in kidney tissue and urine (Loeber and Do¨rries, 1988; Yogo et al., 1990), we attempted to propagate CY in HPT cells (Fig. 2, Table 1). Although Mad1 was shown for the first time to exhibit limited multiplication in this cell type, CY failed to replicate detectably. CY(M1), a chimera expressing CY genes via the Mad1 TCR, was also active in HPT cells, indicat-

TABLE 6 DNA Replication and Viability of Prototype, Variant, and Chimeric JCV in PHFG Cells Experimenta DNA

Day p.t.

1

2

Mad8Br

7 14 21 7 14 21 7 14 21 7 14 21 7 14 21 7 14 21 7 14 21

õ0.1 0.3 õ0.1 õ0.1 0.1 0.3 0.6 0.7 1.1 õ0.1 õ0.1 0.2 õ0.1 õ0.1 0.2 0.3 1.3 7.9 1.0 2.7 24.3

õ0.1 0.2 õ0.1 õ0.1 õ0.1 0.2 0.5 0.6 0.5 õ0.1 õ0.1 0.9 0.4 õ0.1 0.9 õ0.1 2.5 19.2 1.0 12.5 77.2

M1(M8)

M8(M1)

DISCUSSION Multiple genotypes of JCV, BKV, and SV40 have been recovered from human or monkey tissues and during propagation of the viruses in cell culture (Tooze, 1981; Frisque and White, 1992; Ilyinskii et al., 1992). Much of the genomic variability within and between each species has involved rearrangements of sequences that constitute the viral TCRs. Despite this variability, it is clear that multiple isolates of the same viral species are related, prompting suggestions that these variants arose from a common ancestral form. Three such archetype forms have now been identified, one for each of the three primate polyomaviruses (Watanabe and Yoshiike, 1986; Rubinstein et al., 1987; Yogo et al., 1990; Ilyinskii et al.,

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Mad11Br

M1(M11)

M11(M1)

Mad1

Viabilityb

0

{

{

0

{

/

/

a Numerical values for DNA replication were obtained as described for Table 1. b Viability was assessed as described for Table 1.

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ing that minor sequence differences between CY and Mad1 proteins (Loeber and Do¨rries, 1988; Iida et al., 1993) were not responsible for the failure of the parental CY virus to replicate. It should be noted that CY(M1) was less active than Mad1 in renal cells, so one cannot rule out the possibility that CY proteins play some part in archetype’s inefficient replication. We do not believe this would entail a major role, however, since the lytic behaviors of CY(M1) and Mad1 are comparable in cultures of glial cells (see below). Rearranged forms of JCV replicate most efficiently in PHFG cells, so attempts were made to propagate CY in these cells (Fig. 5, Tables 2 and 4). Although CY replicated its DNA to a limited extent in glial cells, infectious virion production was not detected under conditions in which Mad1 and CY(M1) multiplied efficiently. These experiments also confirmed our expectation that M1D98, with a TCR intermediate in structure between that of Mad1 and CY, would demonstrate intermediate replicative activity. Based upon results from the HPT and PHFG cell experiments, we directed our attention toward identifying sequences within the CY TCR that restrict viral replication. Using a series of replication origin constructs and the cell line POJ-19, Mad1 TAg was found to interact productively with the CY origin to drive DNA replication (Fig. 3). By demonstrating that the CY origin as well as CY coding regions were functional, we concluded that the defect in CY replication was due to a failure of the early promoter to express adequate levels of mRNA to support CY TAgmediated replication. Furthermore, CY’s inability to efficiently produce infectious virions, even in cells providing endogenous TAg in trans (POJ-19 cells), indicated the CY late promoter might also exhibit impaired activity (Fig. 4). To compare early and late promoter functions of archetype and rearranged JCV directly, CAT assays were conducted in U-87MG glioblastoma cells (Table 3). The CY TCR induced only low levels of CAT relative to the rearranged TCR; the lowest activity was observed when the CY TCR was joined to the CAT gene in the late orientation. These data support results from the replication studies and indicate that deletion of one copy of the JCV promoter–enhancer and the presence of the 23- and 66-bp sequences have adverse effects on early and/or late promoter function. To identify more precisely the viral sequences contributing to restricted activity of the CY transcriptional apparatus, a series of JCV constructs containing parental, mutant, and chimeric TCRs were created. Our attention focused upon the 23- and 66-bp sequences present in the archetype TCR but absent in the rearranged TCRs of Mad1 and Mad4. Transfection of PHFG cells with the various DNAs indicated that removal of the 66-bp block [M1(CYD66)] elevated replication only slightly above that of CY, whereas removal of the 23-bp block [M1(CYD23)] or the 23- and 66-bp blocks together (M1D98) resulted

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in substantial increases in activity relative to that of archetype (Fig. 5, Table 4). We also observed that CY and M1(CYD66) DNA replication peaked at Day 14 p.t. and tapered off by Day 21 p.t. (Fig. 5). Since secondary JCV infection within PHFG cultures first occurs about Day 11 p.i. (Padgett et al., 1977), the failure of CY and M1(CYD66) to sustain an infection might indicate that the 23-bp block has a more detrimental effect on late promoter function. These observations were supported by a second set of CAT assays showing that both orientations of the CYD66 TCR directed less CAT expression than the CYD23 TCR and that the effect was more pronounced for the late promoter (Table 5). It should be noted that the two CY–CAT constructs were much more active than either the CYD66–CAT or the CYD23–CAT vectors (Table 5). Based upon the replication data (Table 4), we had predicted that the CYD23 TCR would be more active than the CY and CYD66 TCRs. While we cannot resolve this discrepancy at present, it is possible that the JCV promoter–enhancer responds differently in U-87MG cells (commonly used in JCV–CAT assays) and PHFG cells (used in JCV DNA replication and viability experiments). Alternatively, the discrepancy may arise because the two sets of assays measure different biological parameters. In this case we would argue that the replication experiments more closely reflect the complex interactions involved in infectious virion production. Multiple independent isolations of both Mad1 and Mad4 have been made from the lymphocytes and brains of individuals with and without PML (Tornatore et al., 1992; White et al., 1992; Do¨rries et al., 1994), and thus their TCRs may represent preferred rearranged structures. However, most JCV variants isolated directly from PML brain tissue have TCRs which are intermediate in structure between archetype and Mad1; the TCRs contain duplicated regions, lack the 66-bp sequence, and retain the 23-bp sequence. While these rearranged forms of JCV presumably were capable of causing disease in their host, a number of them are replication-defective in cell culture. Two such variants, Mad8Br and Mad11Br, were found in the present study to replicate their DNA inefficiently and to fail to produce infectious virions (Fig. 6, Table 6). Mad8Br, which contains one complete copy and a partial second copy of the 23-bp sequence, was less active than Mad11Br. In addition, its DNA replicating ability, like that of CY and M1(CYD66), peaked at Day 14 p.t. and declined by Day 21. Replacing the Mad8Br and Mad11Br TCRs with that of Mad1 yielded more active genomes; M11(M1) activity approached that of Mad1, while M8(M1) activity was only partially enhanced. This latter observation suggests that a Mad8Br protein(s), in addition to promoter–enhancer sequences, might contribute to the inactivity of this variant. Our data indicate that three features of the archetype TCR influence transcriptional behavior: (i) the absence

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RESTRICTED LYTIC ACTIVITY OF ARCHETYPE JCV

of long tandem duplications, (ii) the presence of a 66-bp sequence, and (iii) the presence of a 23-bp sequence. Numerous studies have already demonstrated the positive effects of tandem repeated sequences on viral promoter–enhancer signals (Hara et al., 1986; Frisque and White, 1992; Lednicky et al., 1995), and the analyses of rearranged (Mad1, Mad8Br, Mad11Br) and mutant (M1D98) forms of JCV in the present study (Figs. 5 and 6) are in agreement with these earlier findings. It was also anticipated that the 66-bp sequence would influence archetype function, since most (but not all) rearranged JCV isolates have suffered complete or partial deletion of this sequence. One possible consequence of these deletions is the loss of a sequence having extensive homology with a negative regulatory element in the BKV enhancer (Grinnell et al., 1988). Our finding that the 23-bp sequence inhibits archetype function was unexpected, since this block of DNA is frequently retained in many rearranged forms of JCV. It is possible that deleting the 23-bp sequence enhances TCR function via the creation of a new cis-acting signal at the deletion boundaries (Markowitz et al., 1990; Johnsen et al., 1995). One such potential signal, the lytic control element (LCE; Tada and Khalili, 1992), has been identified. The LCE affects local DNA structure (Amirhaeri et al., 1988; Chang et al., 1994), enhances JCV TAg-mediated DNA replication (Lynch and Frisque, 1990; Sock et al., 1991; Chang et al., 1994), and binds several transcription factors that affect early and late promoter function (Tada et al., 1991; Tada and Khalili, 1992; Kerr et al., 1994; Kumar et al., 1994; Chen et al., 1995). It is also possible that loss of the 23-bp sequence elevates TCR function due to the removal of a negative cis-acting element (Grinnell et al., 1988; Negrini et al., 1991). The 23-bp sequence does contain a unique binding site for Sp1 (Henson, 1994); however, this transcription factor has not been shown to repress the JCV early or late promoter. Recently, a cross-interaction between the 23-bp sequence and a closely positioned NF-kB motif has been demonstrated, and it was suggested that the interplay of factors binding to these sequences might regulate archetype early and late transcription (Mayreddy, Safak, Razmara, and Khalili, submitted for publication). Such an interaction would likely be relevant to the mechanism(s) by which archetype JCV establishes a persistent infection and is reactivated by changes to the host’s immune or hormonal status. ACKNOWLEDGMENTS We thank John Todd and Donald Sens for adult human kidney proximal tubule cells, and culture medium and protocols for their propagation, and Yoshiaki Yogo for cloned archetype DNA pJC-CY. We also thank members of our laboratory who contributed to the construction of the various JCV chimeras: Patricia MacKeen, John Conway, Mark Hafner, Timothy Sturgeon, and Andrew Yurich. This work was supported by Public Health Service Grants from the National Cancer Institute (CA44970; R.J.F.) and the National Institute of Neurological Disor-

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ders and Stroke (K.K.). A.M.D. was supported in part by a Sigma Xi Grant-in-Aid of Research.

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