Kilham Polyomavirus: Activation of Gene Expression and DNA Replication in Mouse Fibroblast Cells by an Enhancer Substitution

Abstract

The Kilham strain of polyomavirus (KV) infects vascular endothelial cells in vivo (J. E. Greenlee, Infect. Immun. 26:705–713, 1979), but no permissive cell type for growth of the virus in vitro has been identified. The failure of KV DNA to replicate in mouse fibroblast cells after transfection suggested that viral gene expression had narrow cell specificity. A KV substitution mutant having a part of the regulatory region of KV DNA replaced with a segment of the polyomavirus transcriptional enhancer was constructed. The substitution mutant was able to replicate in transfected 3T3 cells, and the newly replicated viral DNA associated with protein to form particles with the density of virions in CsCl equilibrium gradients. However, these particles were noninfectious when tested on 3T3 cells, suggesting that absorption or uptake of virus particles was defective for these cells. Analysis of early and late promoter activities by luciferase reporter gene expression showed that the enhancer substitution had a moderate positive effect on early gene expression and a large effect on the expression of the late genes. KV large T antigen inhibited the activities of both the wild-type and the substitution mutant early promoter, whereas only the mutant late promoter was activated under the same conditions. A comparison of the KV and polyomavirus large T antigens showed that they were not interchangeable in the initiation of KV and polyomavirus DNA synthesis. Furthermore, the wild-type KV origin of DNA replication was less active than the mutant structure in the presence of saturating amounts of KV large T antigen. Together, our data demonstrate several differences between the two types of large T antigen in their interactions with cellular proteins.

The Kilham strain of polyomavirus (KV) is a second murine member of the polyomavirus family, first isolated by Kilham and Murphy in 1953 (31). KV, in contrast to other mammalian polyomaviruses, is associated with severe disease. Infection of newborn mice causes interstitial pneumonia with a high fatality rate (19, 20), whereas exposure of fully immunocompetent mice to KV leads to a persistent and inapparent infection. In primary infection, KV replicates mostly in vascular endothelial cells of the lung, liver, and spleen (19–21, 38). However, during the persistence phase, infected cells are found mainly in renal tubules (24). Thus, although KV and mouse polyomavirus (PyV) show differences in organ and cell tropism during primary infection, they appear to share specificity for tubulus epithelium during persistent infection (22). A major difference between KV and PyV is that inoculation of newborn mice with KV does not result in tumors (23, 41, 43). However, cells transformed with KV can form transplantable tumors (43). Both the virulence upon primary infection of suckling mice and the nontumorigenic phenotype separate KV from PyV, justifying a comparative investigation of the two viruses.

The genome of KV is a circular, 4,754-bp double-stranded DNA molecule. As predicted from the nucleotide sequence (37), KV DNA encodes two early proteins (large and small T antigens) and three late proteins (VP1, VP2, and VP3). Although analysis of cloned DNA definitely established that KV belongs to the polyomaviruses (32), it is not closely related to the previously characterized PyV (3, 37). Unlike PyV and the hamster polyomavirus, KV does not encode any middle T antigen. In this respect and by comparison of deduced amino acid sequences, KV is more closely related to the human polyomaviruses JCV and BKV.

KV exhibits stringent host and cell specificities, and the absence of a permissive tissue culture system has hampered its study. A possible determinant of cell tropism is the enhancer segment of the regulatory region of the genome. With PyV, numerous mutants with altered host range have been described (18, 28, 35). Most of these have point mutations or rearrangements of the enhancer segment, and these mutations have been directly linked to the host range phenotype. Similar observations have been made with the primate polyomaviruses (46).

To investigate the biological properties of KV, we analyzed viral gene expression and DNA replication in mouse fibroblasts and attempted to widen the host range of KV by genetic manipulation of the KV regulatory region.

MATERIALS AND METHODS

Cells and virus.

The mouse cell lines NIH 3T3, BALB/c 3T3, and Swiss 3T6 were obtained from the European Collection of Cell Cultures (Porton Down, England). They were cultured in Dulbecco modified Eagle medium (Gibco) supplemented with 10% newborn calf serum. IE cells, derived from mouse brain capillary endothelial cells (27), were kindly provided by P. Gerwins, Uppsala University, and were grown in Ham F-12 medium supplemented with 10% fetal calf serum and 20 IU of gamma interferon per ml. KV was obtained from the American Type Culture Collection (Manassas, Va.) as a suspension of organ material. Infections were carried out by inoculating cells with virus suspended in Tris-buffered saline (13) for 2 h at 37°C. For transfection, cell cultures were started at a density of 2.5 × 10⁵ per 60-mm-diameter petri dish. On the following day the cells were transfected with 0.3 μg of DNA using the DEAE-dextran method (34) or with 4 μg of DNA using the Lipofectamine procedure according to the instructions of the manufacturer (Life Technologies Products).

Viral genomes and construction of recombinant plasmids.

The recombinant plasmid pKV19, carrying KV DNA in the XbaI site of pUC12 (37), was obtained from Kristina Dörries (Würzburg University, Würzburg, Germany). In this report, pKV19 is called pKVwt. DNA of the wild-type A2 strain of PyV was propagated as a recombinant of plasmid pML2, as described previously (2). PCR was done using the high-fidelity Vent DNA polymerase (New England Biolabs). For cloning of DNA made by PCR, A residues were added to the 3′ ends of the molecules by use of Taq DNA polymerase (MBI Fermentas). The KV mutant KVm1 was generated by replacing part of the pKVwt DNA (XhoI-NarI) with a corresponding region of PyV DNA (nucleotides [nt] 5102 to 5231) synthesized by PCR. Reporter gene plasmids were constructed by ligating polyomavirus promoter DNA segments into the multiple cloning site of the pGL2-basic vector (Promega), which carries the luciferase gene downstream of the cloning site. The early and late promoters of KVwt and KVm1 were synthesized by PCR using pKVwt and pKVm1 DNAs as templates and oligonucleotide primers corresponding to nt 4624 to 4641 and nt 351 to 335, respectively, of KV DNA. The resulting PCR products were cloned into the SacI site of the pGL2-basic vector (pGL2-basic-/KVwtrr, and -/KVm1rr). The corresponding promoter segment of PyV DNA was isolated from pPYE-CAT and pPYL-CAT (1) by XbaI digestion. The resulting DNA fragments were inserted into the NheI site of the pGL2-basic vector (pGL2-basic/PyVrr). KV and PyV large T antigens were expressed by inserting the coding sequence of the protein into the vector pcDNA3 (Invitrogen), which contains the cytomegalovirus immediate-early promoter. The resulting plasmids were designated pcDNA3/KV-LT and pcDNA3/PyV-LT, respectively.

Double-stranded DNA probes specific for KV and PyV DNA were prepared from restriction endonuclease fragments (nt 2094 to 2803 of KV DNA and nt 5102 to 5231 of PyV DNA). A probe for detection of viral origin DNA replication was made by cleavage of pGL2-basic plasmid DNA with HindIII and EcoRI followed by isolation of the smaller fragment (≈600 bp). Radioactive labeling of the DNA from [α-³²P]dCTP was done by random oligonucleotide-primed synthesis (16).

Analysis of viral DNA replication.

Viral DNA replication was analyzed using transfected NIH 3T3 or 3T6 cells. The experiments were carried out with complete viral genomes prepared by excision from recombinant plasmids and recircularization by treatment with T4 DNA ligase at a DNA concentration of 5 μg per ml. Alternatively, replication was monitored using plasmids containing the polyomavirus regulatory region (pGL2-basic/KVwtrr, pGL2-basic/KVm1rr, or pGL2-basic/PyVrr). In these experiments KV or PyV large T antigen was expressed from pcDNA3/KV-LT and pcDNA3/PyV-LT, respectively. Cells were harvested at 42 to 44 h posttransfection. Low-molecular-weight DNA was extracted from the cells, partially purified (26, 40), and cleaved with DpnI and a second enzyme having one cleavage site (SacI for KVwt and KVm1 or BamHI for PyV). Replicated DNA was separated from DpnI-cleaved, unreplicated molecules by agarose gel electrophoresis and was then transferred to a GeneScreen hybridization membrane by capillary blotting, according to the instructions of the manufacturer (NEN Research Products). DNA on the membrane was annealed with ³²P-labeled DNA probes.

Assay of luciferase activity.

Transfected NIH 3T3 cells in 60-mm-diameter dishes were washed twice with ice-cold phosphate-buffered saline and then lysed by the addition of 300 μl lysis buffer (1% Triton X-100, 25 mM glycyl-glycine [pH 7.8], 15 mM MgSO₄, 4 mM EGTA) (6, 39). After incubation for 15 min on ice, cells were scraped off the plates and debris was removed by centrifugation for 1 min at 7,000 × g. Analysis of luciferase activity was done in a Luminoskan luminometer (Labsystems, Helsinki, Finland). Ten-microliter portions of the cell extracts were mixed with 50 μl of a solution consisting of 20 mM Tricine (pH 7.8), 2.67 mM MgSO₄, 1.07 mM MgCO₃, 0.10 mM EDTA, 33.3 mM dithiothreitol, 0.53 mM ATP, 0.27 mM coenzyme A, and 0.47 mM luciferin (Promega). Integrated values for luminescence intensity during 60-s intervals were recorded. In all cases, three or more separate transfections were performed, and the results shown are the average values from the experiments.

Analysis of virus assembly.

Virions and viral nucleoprotein complexes were extracted from NIH 3T3 cells at 40 h posttransfection as described previously (49). The cultures were washed with Tris-buffered saline and then with a low-salt buffer (10 mM HEPES [pH 7.9], 5.0 mM KCl, 1.0 mM CaCl₂, 1.0 mM MgCl₂). The cells were scraped off the plate, resuspended in the low-salt buffer, and then lysed by three cycles of freeze-thawing. Cell debris was removed by centrifugation for 10 min at 10,000 × g, and material in the supernatants was resolved by CsCl equilibrium density gradient centrifugation. Centrifugation was carried out in an SW50.1 rotor (Beckman Instruments Inc.) for 20 h at 35 krpm and 20°C, and fractions were collected from the bottoms of the tubes. The refractive index of each fraction was determined, to assess the density. Each fraction was extracted with phenol and chloroform, and DNA was transferred to a hybridization membrane by dot blotting and annealed with a ³²P-labeled viral DNA probe.

RESULTS

Replication of KV in mouse cell lines and effect of an enhancer substitution.

KV has a restricted host range in vivo and does not infect standard mouse cell lines (37). In initial experiments the susceptibilities of various cell lines, including NIH 3T3 cells, BALB/c 3T3 cells, and the endothelial cell-like IE cells, to KV were tested. The cells were exposed to a virus preparation obtained from lung tissue of infected mice, but none of the three cell lines appeared to be susceptible to the virus. To investigate whether the restriction was at the level of absorption to the cells or penetration into the cells, transfection with KV DNA prepared from the recombinant plasmid pKVwt was performed. As a positive control, PyV DNA was used. In this experiment (data not shown), no newly replicated KV DNA was detected in any of the transfected cultures. Moreover, no infectious KV was recovered from extracts of the cells, as analyzed by serial blind inoculation of cell cultures. At the same time, all of the cell lines were susceptible to transfection with PyV DNA. Newly replicated PyV DNA was easily detectable, and a cytopathic effect was observed in a fraction of the transfected NIH 3T3 and BALB/c 3T3 cultures after 2 days. The results of this experiment suggest that there was an intracellular restriction to KV replication in cells fully permissive to PyV. However, the experiment did not rule out the possibility that the tested cells had an additional restriction in virus uptake.

Many polyomavirus mutants with widened or restricted host range have base pair substitutions or more complex rearrangements of the enhancer segment of the viral genome (18, 28, 35). To investigate the importance of the KV DNA enhancer for the narrow host range of the virus, we replaced part of the structure with a corresponding segment from PyV DNA (Fig. 1). KV DNA was cleaved with the restriction endonucleases XhoI and NarI to remove a 126-bp fragment. A 130-bp fragment of PyV DNA (nt 5102 to 5231) containing the major part of the PyV enhancer was then inserted, and the resulting substitution mutant was designated KVm1.

FIG. 1.

Schematic representation of the regulatory regions of KV and PyV DNAs. In PyV DNA the positions of the replication origin (ori^rep), the transcriptional enhancer core elements A and B, and the 5′ ends of early and late RNAs (10, 45) are indicated. Corresponding positions in KV DNA, as deduced from the nucleotide sequence (37), are also shown. The arrowheads represent large-T-antigen binding motifs (GPuGGC), and the hatched boxes represent a run of A-T pairs at the replication origin. The crosshatched DNA segment indicates the substitution made in KVm1 DNA. Numbers refer to the established nucleotide sequences (37, 42).

To investigate whether the PyV enhancer was able to expand the host range of KV, KVm1 DNA was excised from the recombinant plasmid, recircularized, and used for transfection of Swiss 3T6 cells. PyV DNA and KVwt DNA were used as controls. Low-molecular-weight DNA was extracted at 42 h posttransfection, partially purified, digested with DpnI to degrade unreplicated DNA, and subjected to Southern blot analysis. Since KVm1, KVwt, and PyV DNA do not have any segment in common, the DNA samples were blotted onto two separate membranes and were then analyzed with different ³²P-labeled DNA probes. In mouse 3T6 cells, the KVm1 genome replicated well, while no synthesis of KVwt DNA was detectable (Fig. 2A). Although this experiment showed a positive effect of the PyV enhancer on the KV replication in 3T6 cells, a direct comparison between KVm1 and PyV DNA replication (Fig. 2B) showed that the PyV origin of replication was approximately fivefold more effective under these conditions. The same result was obtained when viral DNA replication was tested in NIH 3T3 and BALB/c 3T3 cells (data not shown).

FIG. 2.

Analysis of viral DNA replication in 3T6 mouse fibroblasts. Rapidly growing cells were transfected with PyV, KVwt, and KVm1 DNAs, respectively, using DEAE-dextran. Viral DNA molecules were prepared by excision from recombinant plasmids followed by recircularization. At 42 h posttransfection, low-molecular-weight DNA was selectively extracted from the cells and partially purified. After digestion with restriction endonuclease DpnI and a second enzyme, making replicated molecules linear, DNA was resolved by agarose gel electrophoresis. Following transfer of DNA to hybridization membranes, it was annealed with ³²P-labeled KV (A) or PyV (B) DNA probes. The membranes were then analyzed by autoradiography. Radioactivity was quantified in a PhosphorImager (C). The positions of linear KV and PyV DNAs visualized by ethidium bromide staining are indicated by arrows.

To investigate whether the replication defect was a result of low large-T-antigen expression, NIH 3T3 cells were cotransfected with recircularized, full-length KVwt or KVm1 DNA (0.5 μg) and a second nonreplicating plasmid expressing large T antigen (0 to 2 μg of pcDNA3/KV-LT). In the absence of large T antigen expressed in trans, KVwt DNA failed to replicate. However, in the presence of the protein, the wild-type origin of replication was functional (Fig. 3A). The stimulatory effect of large T antigen on viral DNA synthesis was also apparent with KVm1 DNA. However, viral DNA replication was increased only with small amounts of cotransfecting pcDNA3/KV-LT plasmid, and quantitation of the hybridization signals showed that there was a complex relationship between viral DNA replication and the total amount of DNA used for transfection. In Fig. 3B the quantity of replicated DNA is expressed as a percentage of the total viral DNA extracted from the cell culture. Calculated in this way, cotransfection of the cells with pcDNA3/KV-LT stimulated viral DNA replication only when small amounts of the expression plasmid were used. A larger input of the helper DNA did not increase, or even inhibited, viral DNA replication. This effect is more likely to be a result of competition between the viral genome and the expression plasmid DNA for intracellular factors than of an inhibitory activity of large T antigen on viral DNA replication. Regardless, the data show that one reason for the failure of KVwt DNA in replication was low expression of the early genes.

FIG. 3.

Viral DNA replication in NIH 3T3 mouse fibroblasts and effect of large T antigen expressed in trans. Growing cells were transfected, using Lipofectamine, with KVwt or KVm1 DNA mixed with the indicated amount of pcDNA3/KV-LT (KV-LT). Extraction and analysis of viral DNA were done as described in the legend to Fig. 2. (A) Southern blot analysis of viral DNA. (B) Fraction of DpnI-resistant viral DNA normalized to the fraction of KVm1 DNA synthesized in the absence of separate large-T-antigen expression.

In spite of the significant replication of KVm1 DNA, the cell cultures showed no cytopathic effect. In addition, no infectious virus was recovered from the transfected cells, as tested on BALB/c 3T3 cultures. The absence of infectious virus might be a result of defective late gene expression, since the replacement of the enhancer region in KVm1 affected the predicted late promoter domain of KV DNA (37) (Fig. 1).

Effect of the m1 enhancer substitution on the activity of the viral origin of DNA replication.

Initiation of polyomavirus DNA replication has a dual dependence on the enhancer segment of the genome. In addition to the expression of the viral initiator protein large T antigen, the initiation event itself requires the activity of an enhancer adjacent to the replication origin (9). To investigate the activity of the KV enhancer in the initiation event, NIH 3T3 cells were transfected with two plasmids. One carried the origin of viral DNA replication but no other elements of viral origin (pGL2-basic/KVwtrr, pGL2-basic/KVm1rr, or pGL2-basic/PyVrr) and served as a reporter of viral DNA replication. The second nonreplicating plasmid encoded large T antigen (pcDNA3/KV-LT or pcDNA3/PyV-LT). The KVwt and KVm1 regulatory regions were tested, and, as a reference, the PyV regulatory region was used. The activities of these three origins of replication were analyzed in the presence of the KV and PyV large T antigens.

NIH 3T3 cells were transfected with 2 μg of plasmid DNA, containing either the KVwt or KVm1 origin of replication, mixed with 0 to 2 μg of pcDNA3/KV-LT. At 42 h posttransfection newly replicated DNA was prepared, and in a Southern blot analysis (Fig. 4A) the hybridization signals of replicated DNA molecules were separated from those of DpnI-sensitive unreplicated DNA. Quantitation of viral DNA replication was done by determining the DpnI-resistant fraction of total viral DNA extracted from each cell culture (Fig. 4B). The data show that saturating amounts of large T antigen were produced from 0.5 μg of pcDNA3/KV-LT and that KVm1 DNA replicated about twice as well as KVwt DNA under these conditions. Thus, the m1 substitution had a direct positive effect on the activity of the replication origin.

FIG. 4.

Activities of the KV and PyV origins of replication with large T antigen expressed in trans. Growing NIH 3T3 cells were transfected, using Lipofectamine, with one plasmid containing an isolated viral origin of replication mixed with a second plasmid expressing large T antigen. (A) Cells were cotransfected with 2 μg of pGL2-basic/KVwtrr or pGL2-basic/KVm1rr, carrying a viral origin of replication (ori^rep), and the indicated quantities of pcDNA3/KV-LT. pcDNA3 without insert was added to give 2 μg of expression plasmid per transfected culture. Low-molecular-weight DNA was prepared and analyzed by Southern blotting as described in the legend to Fig. 2. (B) The hybridization signals of DpnI-resistant and -susceptible DNA were quantified, using a PhosphorImager, and the relative signal intensity of DpnI-resistant material was calculated. (C) Cells were cotransfected with 2 μg of pGL2-basic/KVwtrr (wt), -/KVm1rr (m1), or -/PyV (p) and 2 μg of pcDNA3/KV-LT or -/PyV-LT. As negative controls, plasmid pGL2-basic without a replication origin (−) and pcDNA3 without the large T-antigen-coding region were used. Analysis of DNA and processing of data were done as described for panels A and B. The positions of size markers (in kilobase pairs) are shown on the left.

The activities of the KV and PyV replication origins and the specificities of the two types of large T antigen for their cognate origin structures were also compared. For this purpose, NIH 3T3 cells were transfected with 2 μg of a plasmid containing either the KV or PyV origin and 2 μg of a second plasmid (pcDNA3/KV-LT or pcDNA3/PyV-LT) expressing either KV or PyV large T antigen. The cells were harvested at 42 h posttransfection, and low-molecular-weight DNA was isolated and processed as described above. The results (Fig. 4C) show that the KV and PyV origins of DNA replication were active only in the presence of the cognate large T antigen. Apparently, KV large T antigen did not initiate DNA synthesis at the PyV origin of replication and vice versa. Furthermore, the PyV origin of replication was approximately twice as active as the KV structure, even under these experimental conditions with an excess of large T antigen. Also, in this experiment the m1 substitution had a positive effect on the activity of the KV origin of DNA replication.

Effects of the m1 enhancer substitution on the activity of the early and late promoters.

To analyze the effect of the m1 substitution on promoter activity, the regulatory regions of KVm1 and KVwt were amplified by PCR and cloned in both orientations in a plasmid carrying the luciferase reporter gene (pGL2-basic). As reference material, the corresponding segment of PyV DNA was isolated and cloned in the same reporter plasmid. The nucleotide sequences of the cloned KV DNA segments were analyzed to exclude the possibility that errors had occurred during the PCR.

The reporter gene constructs were transfected into NIH 3T3 cells, cytoplasmic protein was extracted at 40 h posttransfection, and luciferase activity was assayed. The result of the experiment (Fig. 5) showed that the early KVwt promoter was active in NIH 3T3 cells but that its activity was about half of that obtained with the early PyV promoter. In contrast, the late KVwt promoter had a very low activity, both in absolute terms and in comparison to the PyV late promoter. The m1 substitution in the enhancer had effects on both the early and late KV promoters. The strongest effect was on the late KV promoter, which was activated more than 30-fold, almost to the level of the late PyV promoter. At the same time, the m1 substitution appeared to decrease the activity of the early promoter approximately threefold, which was unexpected considering its positive effect on DNA synthesis.

FIG. 5.

Analysis of early and late promoter activities by reporter gene expression. Growing NIH 3T3 cells were transfected, using Lipofectamine, with 4.0 μg of plasmid pGL2-basic/KVwtrr, -/KVm1rr, or -/PyVrr with the regulatory regions in the early (E) or late (L) orientation relative to the luciferase reporter gene. Cytoplasmic protein was extracted at 40 h posttransfection, and luciferase activity was assayed in duplicate samples. Transfections were carried out in triplicate, and the variation in luciferase activity is indicated by error bars.

In the experiment described above, the luciferase reporter gene expression was analyzed in the absence of large T antigen. However, viral DNA replication occurs only in the presence of this protein. Therefore, the reporter expression experiment was carried out in cells expressing large T antigen. In this experiment, NIH 3T3 cells were cotransfected with the luciferase reporter constructs and pcDNA3/KV-LT or pcDNA3/PyV-LT. As in the experiment described above, the KVwt, KVm1, and PyV regulatory regions were tested in the early and late orientations. All transfections were done with the same quantities of plasmid DNA, and as a negative control, pcDNA3 without a large-T-antigen-coding sequence was used.

The luciferase expression driven by the early promoters was lower in cells cotransfected with the plasmid derivatives encoding large T antigen or the pcDNA3 control (Fig. 6A) than in the absence of the expression vector (Fig. 5). Part of this inhibition was probably a result of competition between the polyomavirus and cytomegalovirus promoters for transcription factors. However, there was an additional negative effect on the early promoters by large T antigen, as demonstrated in earlier studies (1, 15). KV and PyV large T antigens inhibited the activities of all three types of early promoters, although with somewhat different efficiencies. For the KV early promoter, the m1 substitution improved its performance (Fig. 6A), which is different from the effect of the substitution in the absence of large T antigen. Analysis of the late promoters gave a different result (Fig. 6B). Like in the absence of large T antigen, the KVwt late promoter had a very low activity in NIH 3T3 cells, and this activity was not increased by KV or PyV large T antigen. In contrast, both the KVm1 and PyV late promoters were transactivated by large T antigen. KV and PyV large T antigens had similar stimulatory activities on the KVm1 late promoter, while the PyV late promoter was specifically and strongly stimulated by PyV large T antigen.

FIG. 6.

Effect of large T antigen on viral early (A) and late (B) promoter activities. Growing NIH 3T3 cells were transfected with 2.0 μg of DNA of plasmid pGL2-basic/KVwtrr, -/KVm1rr, or -/PyVrr, with the insert in the early (E) or late (L) orientation relative to the luciferase reporter gene, and 2.0 μg of pcDNA3/KV-LT or -/PyV-LT. As a negative control, pcDNA3 without insert was used. Cytoplasmic protein was extracted at 40 h posttransfection, and luciferase activity was assayed. The inset in panel B shows the activity of the KVwt late promoter in an expanded scale. Error bars indicate the extreme values.

KVm1 virion assembly in transfected cells.

Since KVm1 replicated in mouse NIH 3T3 cells and appeared to express the late genes, we examined whether virus particles were formed. NIH 3T3 cells were transfected with KVm1 DNA prepared from recombinant plasmid DNA, and as controls, KVwt DNA and PyV DNA were used. Cells were also cotransfected with KVwt DNA and pcDNA3/KV-LT to examine whether virions were formed once viral DNA had been synthesized. At 42 h posttransfection, the cultures were harvested by extraction of cells under conditions for release of virions, and the cell extracts were applied to CsCl density gradients. After centrifugation, the gradients were fractionated and each fraction was extracted with phenol and chloroform. DNA was dot blotted to a hybridization membrane and annealed with a ³²P-labeled DNA probe. In Fig. 7, the relative amount of viral DNA in each fraction is plotted against the calculated density.

FIG. 7.

Virion formation in transfected NIH 3T3 mouse cells. Growing cells were transfected, using Lipofectamine, with 0.3 μg of PyV (A), KVm1 (B), or KVwt DNA in the presence of 2.0 μg of pcDNA3/KV-LT (C). Viral DNA was prepared by excision from recombinant plasmid followed by recircularization. Transfected cells were harvested at 42 h posttransfection by extraction in low-salt buffer and freeze-thawing three times. Cell debris was removed by centrifugation, and each cell extract was applied to a CsCl density gradient. After centrifugation, the gradients were harvested by collecting fractions from the bottoms of the tubes. The refractive index was measured to determine the CsCl density, and each fraction was extracted with phenol and chloroform. DNA was dot blotted to hybridization membranes, and after annealing to ³²P-labeled DNA probes consisting of PyV or KV, respectively, the hybridization signals on the filters were quantified using a PhosphorImager. The relative value of each signal was plotted against the calculated CsCl density.

In a CsCl density gradient, polyomavirus particles will band at a density of 1.34 g/cm³ (49). The material from cells transfected with PyV DNA formed two bands, one at 1.34 g/cm³ and a second at 1.39 g/cm³ (Fig. 7A), containing virus particles and incompletely packed viral DNA, respectively. Viral DNA from extracts of KVm1-transfected cells had similar banding properties in CsCl, with a peak at a position corresponding to virions (Fig. 7B). However, no such material was extracted from KVwt-transfected cells (data not shown). Interestingly, in cells cotransfected with KVwt and pcDNA3/KV-LT, there was newly replicated DNA but apparently no assembly of virus particles (Fig. 7C). This result is consistent with the idea that a major restriction of KVwt replication in 3T3 cells is weakness of late gene expression, leading to failure of virus formation.

DISCUSSION

In experimental infection of mice with KV, primary replication occurs in vascular endothelial cells in various tissues. During the initial phase of the infection, the largest amounts of virus are recovered from lung, liver, and spleen (19, 20, 38). Similar tissue distributions of KV are observed after oral, intraperitoneal, and intrathecal inoculations, raising the question of what cells are the first to become infected at the portal of entry and how virus is spread from that site. Experimental infection of newborn mice with PyV has a different outcome. This virus replicates in many different cell types and can be recovered from most tissues during the initial phase of the infection (12). The other fundamental difference between KV and PyV is in the ability to induce tumors. The tumorigenicity of PyV has been related to the efficiency of replication in various organs (11). The tissue tropism of KV and PyV in vivo is reflected by the infectivity of the viruses in vitro. Hitherto, no permissive cell culture system for KV has been reported. In contrast, PyV multiplies efficiently in a large number of cell lines and primary cell types in culture. There are no reports on whether the resistance of cultured cells to KV is a result of failure in uptake of virus or in its intracellular replication. Given the relatedness of KV and PyV, the factors determining the host cell specificities and the tumorigenicities of the two viruses pose an interesting problem.

Following initial unsuccessful attempts to isolate KV or newly replicated viral DNA from various mouse cell lines inoculated in vitro, KV DNA was delivered into cells by transfection. Again, KV failed to replicate in mouse 3T3 and 3T6 cells and in the vascular endothelial IE cell line. Under the same conditions, PyV replicated well. Since the failure of KV to grow in transfected cells could not be explained by the absence of a specific receptor located in the plasma membrane, other possibilities had to be considered. In both murine and primate polyomaviruses, the enhancer segment of the genomic regulatory region determines cell-specific gene expression and viral DNA synthesis (12, 46). Therefore, a segment of KV DNA at the presumed position of the enhancer (nt 1 to 124) was replaced with a corresponding segment of PyV DNA known to contain most of the enhancer elements active in fibroblast cells (Fig. 1). This substitution (m1) gave KV DNA the capacity to replicate in mouse 3T3 and 3T6 cells, albeit not to the same level as PyV DNA (Fig. 2).

In PyV and simian virus 40, the enhancer has been shown to have a dual effect on the initiation of viral DNA replication (9). In addition to activation of the early genes, leading to expression of replicator protein (T antigen), transcription factors binding to the enhancer appear to facilitate the initiation step of DNA synthesis (44). Recruitment of large T antigen to the origin of replication is probably promoted by transcription factors in complex with the adjacent enhancer. In PyV DNA synthesis, the transcription factor AP1 (PEA1) has been demonstrated to have this function (25). AP1 is one of many transcription factors with binding sites in the segment from nt 5102 to 5231 of PyV DNA that was used to construct the substitution mutant KVm1. The segment from nt 1 to 124 of the regulatory region in KV DNA, which was deleted in the KVm1 mutant, in addition contains a considerable number of predicted (48) transcription factor binding sites, e.g., recognition sites for Oct1, SP1, NF1, and Ets1. Closer to the origin of replication, not affected by the KVm1 substitution, there are recognition sites for AP1, NF1, and AP4. Whether any of these transcription factor binding sites is functional has not been tested. We also noticed binding sites for the erythroid cell-specific transcription factors GATA C and GATA1 at nt 89 in KVwt DNA. Based on this observation, we tested whether KVwt DNA was able to replicate in the Friend erythroleukemia mouse cell line clone 707. However, the result was negative.

To analyze the effect of the m1 substitution on the activity of the replication origin in a situation independent of early gene expression, cells were transfected with one plasmid containing the KVwt or KVm1 origin of replication and a second plasmid expressing large T antigen at a high level under the control of the cytomegalovirus immediate-early promoter. Under these conditions, both the KVwt and KVm1 origins of replication were active, but the m1 substitution increased the DNA synthesis approximately twofold in the presence of saturating amounts of large T antigen (Fig 4B). However, even at a high large-T-antigen concentration, the KVm1 origin of DNA replication was much less active than the corresponding PyV structure driven by the PyV large T antigen (Fig. 4C). Although the KV and PyV large T antigens activated their cognate origins of replication, there was no cross-reactivity. Both proteins bind to GRGGC motifs organized in tandem (8; S. Zhang and G. Magnusson, unpublished data). However, the organization of these motifs at the origin of replication is different in KV and PyV DNAs, with four pentanucleotide motifs in PyV DNA but only three in KV DNA (Fig. 1). Hence, formation of large-T-antigen hexamers at the origin of replication and subsequent unwinding of double-stranded DNA (14) might differ slightly for the two proteins.

The ability of KVwt to replicate when large T antigen was expressed from a separate plasmid (Fig. 3) suggested that low activity of the early promoter in mouse fibroblasts was a major reason for the defective viral DNA synthesis. However, in combination with an origin of DNA replication having a limited sensitivity to large T antigen, a low early gene expression apparently was detrimental to viral DNA synthesis.

Since the analyses of viral DNA synthesis did not yield any information on the expression of the viral late genes, the activities of the early and late KV promoters were assayed in transfected NIH 3T3 cells using reporter gene constructs. In the absence of large T antigen, the KVwt early promoter had approximately half the activity of the PyV early promoter (Fig. 4). In contrast, the KVwt late promoter was nearly inactive under these conditions. The m1 substitution had opposite effects on the activities of the early and late promoters. The early promoter was inhibited 2.5-fold, whereas the late promoter was stimulated approximately 30-fold. Analyses of the late promoters in PyV and simian virus 40 have not demonstrated well-defined essential DNA elements (8). Hence, it is possible that the segment from nt 1 to 124 of the KVwt regulatory region contains a component having a negative effect in mouse fibroblasts that is absent in the KVm1 mutant genome. A similar phenomenon has been observed with simian virus 40 (47).

We had also expected a positive effect of the m1 substitution on the activity of the early promoter. Hence, the possibility of a different effect of the m1 substitution in the presence of large T antigen was considered. In the analysis, the synthesis of large T antigen was uncoupled from the early KV promoter by cotransfecting the cells with the reporter gene construct and a separate expression plasmid encoding large T antigen. In agreement with earlier observations on the autoregulation of the PyV promoter (7), the presence of either KV or PyV large T antigen in the NIH 3T3 cells inhibited the KVwt, KVm1, and PyV early promoters, although with different efficiencies (Fig. 6A). The inhibition was probably mediated by binding of large T antigen to GAGGC motifs located adjacent to the 5′ ends of the early transcripts (1, 15). In a comparison of the KVwt and KVm1 early promoters, the m1 substitution alleviated the inhibition by large T antigen 1.5- to 2.0-fold. This decreased sensitivity to large T antigen fits with the observed positive effect of the substitution on viral DNA synthesis.

The late PyV promoter is transactivated by large T antigen (4, 5, 29, 30). A four to fivefold activation was also observed in our experiments (Fig. 6B). In contrast, the KVwt late promoter, having a very low activity in the absence of large T antigen, was further inhibited by both the KV and PyV proteins. This trans-repressive effect supports the notion that NIH 3T3 cells contain a factor with a negative activity on late KV transcription. The m1 substitution relieved the negative effect and provided a target for transactivation by large T antigen of the late promoter. However, the approximately threefold transactivation was significantly lower than that for the PyV late promoter. The observation that PyV large T antigen transactivated both the PyV and KVm1 late promoters, whereas the KV large T antigen failed to activate the PyV late promoter, suggested that the two proteins mediated their effects by interaction with different cellular transcription factors.

The final question we addressed in this study was whether virus particles are formed in mouse fibroblast cells by the KVm1 mutant. Since these mutant KV genomes replicated and had an active late promoter, they were expected to express the late genes. Analysis of late RNA in NIH 3T3 cells confirmed that there was cytoplasmic viral RNA at a late time point after transfection with KVm1 DNA (data not shown). Thus, capsid proteins were probably synthesized in the transfected cells. To detect the formation of virus, material extracted under low-salt conditions was resolved by centrifugation in CsCl density gradients (49). To obtain a sufficient sensitivity of the analysis, the DNA component of virions was examined. Extracts of cells transfected with KVm1 and PyV DNA contained material with a density of 1.34 g/cm³ (Fig. 7), which is typical for virus particles. Thus, KV capsid protein and DNA appeared to assemble into virus particles in NIH 3T3 cells. When cells were cotransfected with KVwt DNA and a plasmid expressing KV large T antigen, viral DNA was formed but was not properly encapsidated, since the DNA-containing band was found near the bottom of the gradient. However, the density in this region was considerably less than the banding position of DNA in CsCl (approximately 1.70 g/cm³). Thus, the KVwt DNA might be associated with procapsid structures. Together, the data show that although replication of KV DNA occurred in the presence of large T antigen, virus particles failed to be assembled, probably because of low late gene expression and a consequent lack of capsid proteins.

In spite of the apparent assembly of KVm1 virus, no cytopathic effect was found in the cells, even after extensive incubation to allow for repeated infection cycles. There are several possible reasons for this result. Virus particles formed in NIH 3T3 cells might be noninfectious from a defect in maturation, or the released virus might not be able to reinfect mouse fibroblast cells due to a block in the absorption or uptake processes. In particular, the receptor for KV has not been identified and might not be present on any of the cell types we have tested. Studies on cellular receptors for several other polyomaviruses (17, 33, 36) suggest that they have distinct specificities. We are currently extending studies on the involvement of cellular receptors in the cell specificity of KV.